starch gel electrophoresis of conifer seeds: a laboratory manual

8/8/2019 Starch gel electrophoresis of conifer seeds: a laboratory manual

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United StatesDepartment ofAgricultureForest Service

General Technical


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-TH ,a geneticist, is assigned to the Station's research unit

dyin etics of western forest trees, with headquarters in Berkeley, Calif. Heearned a bachelor's degree in forestry at Michigan State University, a master's degreein forest genetics at North Carolina State University, and agenetics at the University of Ca lifornia, Berkeley. PAUL D.cal technician with the genetics research unit, i sa forestry graCaliforn ia, Berkeley. LUCY .NUNNALLY, a biological technician at the Southeast -ern Forest Experiment Statio n, Raleigh, N.C., earned a bachelor's degree in biology atVanderbilt University. SERENA C. HUNTER received a bachelor's degree in forestryat the University of the South. At the time of this study, she was a graduate student atNorth Carolina State University, where she received a master's degree in forestry.

The procedures described in this manual were adapted from starch gel techniques inuse at the University of California, Davis. Alex Kahler, now plant geneticist, Nort hernGrain Insects Research Laborato ry, Agriculture Research Service, U.S. Depart mentof ~ ~ r i c u l t u r e ,rookings, S.D., provided in formation on electrophoresis t hat led tothe establishment of an isozyme laboratory in 1970at the Pacific Southwest Forest andRange Experiment Station under the direction of M. Thompson Conkle and to thedevelopment of a user's manual . The first draft of the manual was prepared by Lucy B.Nunnally and Serena C . Hunter. Recent modifications of techniques and additions ofstain systems prompted Paul D. Hodgskiss to revise and enlarge the manual. Demandfor copies of the first draft a nd requests for details abou t the procedures encouragedthis publication. Because these procedures are constantly being modified, the authorswould appreciate receiving suggestions for the improvement of this manual.


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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .on . . . . . . . . . . . . . . . . . . . . . . . . . . .rac t ion , and Sto rage ................................................tratification . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . . . .. . . . . . . .erminationparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2on . . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .ta rch

ration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Gel Loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Electrical Requirem ents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

nning Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5..............................................licing the Gel 5ixing the Stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Storing the Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6o n s . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Safety . . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . .. . . . . . . . . . . .. . . . . .Procedural Cautions . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . . .. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Extraction Buffer. Gel and Tray Buffer F ormulations and Electrode Tray

Dim e n s io n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3C. Stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ID. Chemicals Needed for Processes Described. ................. 17

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


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he identification of different form s of enzymes by theprocess of electrophoresis is a pow erful research too le genetic analysis of forest trees (Feret and Bergm ann1976). Electrophoresis is the mo vemen t of enzymes in a gel,under the influence of an electric current . Dissimilar

enzymes and the al ternat ive forms of s imilar enzymesmigrate at different rates when direct current is appl ied toma cerate d seed samples in a buffered sta rch gel. Enzymemigration in gels and the separation of enzymes withdifferent charge s results from the interact ion of the electriccurren t , the pH in the gel, and the pH in the e lec t rode t ray(gel system). Th e direction-anodal or cathodal-and rateof enzyme migration depend on the kind of electriccharge-plus or minus-and the charge strength.Several gel systems are needed to analyze maximumnum bers of enzym es. After gels have been held fo r a suffi-cient time in the electric field, gel slabs ar e sliced an d th ingel slices a re stained for different enzymes. Sta in solution scontain specific substrates , and enzymes from the seedsamples act on these substances to produce react ion pro-ducts . React ion products are s tained or are the bases ofstain processes. The locations of visible band s o n the gelsma rk the m igration distance s of specific enzymes. Differ-ent ban ds on a gel that has been s tained for one react iondeno te functionally related m olecules that differ in electri-cal charge-isozymes. Isozymes, tested by deter minin gMendelian segregat ion rat ios and foun d to be phenotypicexpressions of alleles of a single genetic locus, ar e allozymes.The electrophoresis of conifer seeds has many usefulappl icat ions. Conifer seeds are excel lent for genet ic s tudiesbecause gene products are present in haploid and diploidt issues. The nutrient material surrounding the embry o ishaploid. Electrophoresis of this tissue resolves enzymealleles identical with the fem ale gam ete of the seed. An alysisof gametophytes fr om several seeds of one t ree provides anaccu rate evaluation of that tree's genotype . Electrophoresisof embryos resolves diploid banding patterns. The pollencontribut ion to individual embryos can be deduced bysubtract ing the al lele carried by the game tophyte from thepair of alleles carried by the em bryo .

This manual provides detai led information on seedpreparat ion techniques, equipment , and chemical form u-las , including how to prepare t issue an d gel , supply electriccurre nt , and s tain gel s lices to produ ce vis ible bands m ark-ing the location of enzymes. It outlines proven proce dure sfor obtaining isozyme phenotypes in 23 enzyme systems,for resolving genotypes at a bou t 45 loci. The proceduresfo r conifer seed electrophoresis are presen ted in sections inthe order they are performed during day-to-day lab oratoryoperat ion. This manual , which updates an d supersedes an

earlier publication (C onkle 1972), doe s not cove r geneticinterpr etation of the bands o n the gels.

Seed processing-collection, ext rac tion , storag e, strati-fication, and germination-varies by species (consult Agri-cu l tu re Handbook 450 [U.S. Dep. Agric., Forest Serv.19741 for species inform ation). T he meth ods we describeprovide satisfactory results for a wide range of conifers andare suited for small quantities of seed.Seeds, once col lected and dried, should be frozen forstorage. In this state they rema in viable for man y years andretain high levels of enzym e activity. The efficient schedul-ing of studies using stored seed tissues permits a con stantou tpu t f rom the l abora tory and suppor t s t af f , wi thoutbreaks caused by seaso nal lack of research m aterial.The seeds should be germinated under uniform condi-t ions and sampled a t uniform growth s tages. This proce-dur e guaran tees th at a specific set of enzymes will be activein all seed of the test, an d bypasses pro blem s enco unter edwith tissues sam pled fro m field trees-differences arisingfro m microsites, seasons and y ears, and difficulties of col-lection, transportation, storage, and processing.It is advisable to analyze seed at the stage of germ inationwhen the emerging radicle of the embry o extends 2 to 5 rnmbeyond the seed coat. Enzymes a re present from dr y seedstage through the shed ding of the seed coa t (Conkle 1971),but female gam etophytes an d embryo s in the early stages ofgerminat ion are superior to dry seed for most species .Additionally, germination simplifies removal of the seedcoat an d separat ion of the embryo from the gametophyte.

Collect matur e cones, while still closed, from the tree. A irdry or wa rm the cones in a circulat ing air oven o r ki ln (32OC for 24 to 48 hours). S hake o r tumble the open cones torelease the seeds.Rem ove the seed wings by gently rubb ing a small quan -tity of seed within a folded cloth. Separate th e seed fromthe wing particles bya. Sha king the seed on a wire mesh. Small wing pieceswill sift through the mesh; larger pieces can be removedfrom the seeds by a quick downw ard mo tion of the mesh.


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ng the seed material in a variable-flow airace the seeds and wing fragments on a wire meshinside an air tube a nd increase the air f low unt i l the wingpieces are blown into the collector. This procedure is alsouseful for separating filled from hollow seed.

Sto re dry seeds in labeled envelopes in a freezer (- 15O C).

Withdraw from cold s torage the seed samples to beincluded in a study. P lace seeds of each sam ple in plasticbags labeled with identifying codes. Pour enough Captanfungicide solution (2.5 g Ca ptan 50- I liter of water) intoeach bag t o cover the seeds. ' S oak seeds for 24 hours in arefrigerator (thro ugh out this manua l, refrigeration impliesa temperature of 4" C). Drain the Captan solut ion andrefrigerate the moist seed 90 days for w hite pines an d 45days for a ll other species.Pre par e petri dishes by addin g a cellulose pad or sterilesand. Overlay the pad or sand wi filter paper labeledindelibly with identifying codes. the pad an d filterpaper with Captan solut ion. The fi l ter paper should besaturated but the dish should not have freestanding solu-tion. Transfer th e seeds into the p etri dishes and place in agerminator. Throughout the subsequent germinat ionperiod add distilled water as needed to ma intain mo istureon the seeds and paper.

Germ inate seeds at 20' to 22" C with 12 hours of light.Mos t seed lots begin to germinate in 3 to 7 days. W hen theradicle of a seed extends beyond the seed coat 2 to 5 mm,place the germinant in a second petri dish (prepared withmoist pad or sand a nd fi l ter paper) and refrigerate. R efrig-eration halts the growth of seeds while maintaining enzym eactivity 6 to 8 weeks and longer, and al lows you to accum u-late seeds at the sam e stage of development.

Prepa re a record sheet (fig.I) isting in ord er the identify-ing codes and tissue types for samples to be analyzed.Recom mendation: In add i t ion to the seeds that are beingtested, stratify, germinate, an d include seeds of alternativespecies or particular families as standards for comparingthe mobility of enzyme bands on different gels. Labelgrinding plates (fig. I ) with sample identities correspond-ing with the record sheet.*

'Trade names and commercial enterprises or products are mentionedsolely for information. No endorsemen t by th e U.S. Department of Agri-culture is implied.

*Figures are found on pages 8 to 11 of this manual.

Each sample consists of a paper wick (12 x 3.5 m m ,Whatman chromatography paper, no. 3 M M ) saturatedwith liquid derived from m acerated tissue combined withextraction buffer solution. To obtain the liquid from thegerminated seeds, proceed as follows:a. For each seed, spli t and remove the seed coat and peeloff the brown papery tissue surrou nding the gametophyte.Spl i t the gametophyte on one s ide and remove the em bryo.b. Place the gametophyte t issue and embryo in separatelabeled wells of a frozen grinding block (fig. I). Theamount of tissue to process depends on seed size andenzyme activity. Use the entire gametop hyte o r embry o ofspecies with small seeds but subsample the tissues fromspecies with large seeds.c. Process the samples loaded in the grinding blockimmediately if electrophoresis run will be made th at day.Otherwise, store them frozen overnight so as to reduce theworkload on the day of the run.d. On the day of the run, remove the samples from thefreezer. Add o ne or two dro ps of extraction buffer (app.A)to the tissue in each well. Use the minimum qua ntity neces-sary to just saturate paper wicks; too much ex tract ionbuffer dilutes the enzyme solution an d diminishes the reso-lution of isozyme bands. N ote tha t in appendix A there aresuggestions for additives that improve resolution of spe-cific enzymes, and of enzymes of seeds with large amo un tsof resins.e. Grind thawed, cold tissues in the extraction bufferwith a glass rod o r blunt-tipped electric engraver. Macerateeach sample thoroughly-the mac erate in each well shouldbe opaq ue and contain no sample chunks. After grindingeach sam ple, clean the grinding t ip with abso rbent t issuepaper. Once a sample is homogenized, enzymes begin tobreak down. Work rapidly once you begin the grindingstep.

f. Insert paper wicks in the wells contain ing the macer-ated tissue. Use one wick per well for each gel system t o berun; i f , for example, each sample is to be used in fourdifferent gels, use four wicks per well. Keep the saturatedwicks cold. You can also include on a gel one or m ore wickswith dye marker (0.1% solution bromophenyl blue, 0.1mg/ 100 ml of water) to monito r freely migrating moleculesduring the electrophoresis run.

Four gel systems, with pH values from 6.2 to 8.8, areuseful for resolving different enzymes. T he specific chemi-cal formulations for gel and tray buffers are given inappendix B. Instructions call for preparing two gels of eachgel system. Two gels provide analyses for n ume rous tissuesamples each day and make efficient use of space andmaterials by allowing staining of two slices in each stain


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tray. You may wish to adjust the formulations to meetdifferent experimental design goals.Starc h, when cook ed with a buffer solution and cooled,form s a gel. The p roportio n of s tarch to buffer determinesthe consistency of the gel. Gels tha t are 12.5 percent sta rchappear to work well, but the proportions may need adjust-ing to produce gels with good characteristics. A majorvariable is the bra nd of starch. Othe r factors-the qualit yof dis til led or deionized water , s tarch lot, lab orator y andrefrigerator tem peratures , and the length of t ime th at gelsare vacuum degassed-contribute to gel quality. The re isan a rt to pro ducin g consistently good quality gels; a visit toan electrophoresis laboratory is highly recommended toanyone planning to establish a new laboratory.

Form the gel in a m old consisting of plastic bars securedto s ingle-s trength glass with rubber bands ( fig. 2). Thismold forms a gel slab, 18.5 15.5 x 1.2 cm, capable ofyielding u p to 10 gel slices. Lines o n the side bars serve asguides for cu tting the gel where the wicks will be inserted-a location on the gel called the origin because enzymemigration s tarts from this point. Lines on s ide bars alsomark locations for folding the plastic wrap cover, placingelectrode sponges, and determining the 8-cm migrationdistance for gels that develop a visible front as electro-phoresis proceeds.

Prepare and level two molds for each batch of s tarch.Weigh ou t 94 g of starch. If necessary, remove an y small,hard-to-suspend aggregates by sifting or screening the drystarch. Measure out 750 ml of gel buffer solution fromlabora tory s tock bottles (app .B). Of the 750 ml of solutio n,reserve 160 ml for suspending the s tarch, and heat therem aining 590 ml to bo iling in a 1000-m1 long-neckedvolumetric flask (Pyrex no. 5600). While the solution isheating, use a 400-ml bea ker to com bine 150 ml of thereserved buffer solution with the starch. M ix the starch andbuffer thorou ghly with a stirring rod t o eliminate all lumps.Pour the starch suspension into a 2000-m1 thick-walledvacuum flask (Pyrex no. 5340). Wash the remaining starchfrom the be aker into the vacuum flask with the remaining10 ml of buffer. Swirl the mixture to keep the starch wellsuspended. Here an d elsewhere in the text, the word swirlisused to describe the required m otion fo r the f lask contain-ing the s tarch suspension. Hold your forearm stationaryand use wris t action to produce a circular motion of thebase of the vacuum flask. The s tarch suspension shouldcircle the bottom of the f lask; i t should not break up an dsplash against the wall.Pu t on insulated gloves to handle ho t glassware and w eareye protection. Short ly after the 590 ml of solution comes

to a rap id boi l , g rasp the vacuum f lask in one hand and thevolumetric flask in the other. Swirl the starch in thevacuum flask until the s tarch is again thoroughly sus-pended. T hen carefully invert the volumetric flask conta in-ing the boiling buffer, sliding its neck dee p into the va cuumoint the neck of the vacuum flask away fromyou. CAUTION: Failure t o insert the flask with boilingbuffer deep int o the vacu um flask ma y result in a violentkickback of s team and boiling buffer . Swirl the vacuumflask as the buffer solution empties into the s tarch suspen-sion. Remove the volumetric f lask, when emp ty, and con-tinue swirling the starc h and buffer fo r 10 to 15 seconds.If gels are consistently weak an d difficult to work with,you may need t o heat briefly the suspended sta rch after theboiling buffer is added t o further cook the mixture. Su chfurther cooking may be required to produce gels suffi-ciently strong to withstand slicing and handling.Stopper the f lask and apply vacuum to degas the hotstarch solution (fig. 4). A rapid effervescent boiling willoccur within the s tarch solution as gases are removed, butwill quickly su bside . Slowly swirl the flask while degassingto m aintain a n even consis tency of the s tarch. Continuedegassing until large bubbles boil through the solution.Abo ut 4 seconds after the large bubbles ap pear, release thevacuum a nd imm ediately pour the s tarch into the molds.The st arch solution w ill be clear and will pou r freely. Theprocess of degassing shou ld last only 15 to 20 seconds; thestarch w ill congeal inside the flask if degassing is prolo nged .Rapidly pou r the s tarch into the molds, f il l ing each moldwith one continuous p our, a s the s tarch cools quickly. Star tpouring midway between the upper left corner and thecenter of the mold. W hen the starch reach es the edge of themold a nd m ound s slightly above the top of the plastic bars,continue to po ur, moving to the r ight, then down, then left.Continue pouring until the mold f il ls and s tarch bulgesabo ut 1 mm above the top of the forms. Pour the second gelin the same manner. W ith practice, you can produce twogels with equal quantities of starch, and thus equal thick-ness , from one s tarch preparation.

The s tarch m ixture should be free of opaqu e s treaks andsmall opaque pellets. These opa que areas are usually d ue toincomp lete mixing and coo king when the boiling buffer isadded. If you see inclusions, discard the contents of theflask and sta rt over.After pouring, gently shake or lightly tap each mold todis tr ibute the s tarch evenly. Burst an y bubbles o n the gelsurface with a w arm needle. Cool the gels at room tempera-ture until they turn op aque (ab out 10 to 15 minutes), thencover them w ith plastic wrap. If gels are prepared th e daybefore they are used, s tore them a t room temperature.

Refrigerate prepared gels for 1 hou r before insertingwicks. Never freeze gels. Use a scalpel (no. 11 blades areuseful) to cut an d tr im the gels . Cu t between the gel and all


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four sides of the gel mold. Remove an y excess starch fromthe top of the bars. With the scalpel held ver t ically, cutalon g a straight-edge held in l ine with the or igin mark s onthe right an d left sides of the mold (fig. 2).Remove the rubber bands holding the mold bars andslide the smaller cut portion of the gel toward the edge ofthe glass, using l ight pressure f rom both hands on thesurface of the gel. Th e gel slice should be moved until theopening at the or igin is abo ut 1 cm. Place a wick spacingguide on the up per gel slice ust at the or igin ( fig.5).Repea tthe process to tr im, open , and place wick spacers on thosegels required for the n um ber of wicks prepared.Use tweezers to remove the cold wicks fro m th e wells ofthe grinding block, w orking with on e well at a time. Lightlyblot the wicks on a clean section of absorb ent tissue. Placethe wicks, on e per gel system, on the fresh-cut gel surface ofthe larger gel port ion. Th e bo ttom edge of each wick shouldtouch the glass of the gel mold. The order of wicks alongthe gel should correspo nd to the sam ple order o n the recordsheet. Each gel acco mm oda tes 36 wicks.When all the wicks are in place, push the smaller gelport ion back against the wicks on the larger gel port ion.Replace the gel mold bar an d ru bbe r bands. Press lightlyon the gel near the wicks to remove air gaps. C over the gelwith plast ic wrap tha t extends abou t 5 cm beyond the endsof the mold.

Place the loaded gel on the electrode tray (seefig. 6 fo rdetai ls of tray con struction) . Posit ion th e or igin of the geltoward the cathodal connection of the electrode tray.Form ing a sh arp l ine across the gel , fold the p last ic wrapback to the fold l ines ( fig. 2) marked on the gel bars. Thisprocedure exposes an 18-mm strip of gel surface.Place sponges in contact with gel surface, in each elec-trode tray. The spo nges are nylon reinforced cellulose kit-chen sponges. Because new sponges increase the t imerequired for an electrophoresis run, co ndit ion new spongesby r insing and using them in ano dal trays with previouslycondit ioned sponges in catho dal trays. D o not interchangethe spong es between systems. Sto re sponges in plastic bagsin a refrigerator.Posit ion sponges to cover 1 cm of plastic and all theexposed gel surface, and extend down into the electrodetray. Satu rate sponges with electrode tray buffer and presssponges against the gel to m ake thoro ugh co ntact . Fold theplast ic wrap back over the sponge and at tach the plast icwrap to th e glass on both sides with binder cl ips (seef ig. 7for views of the tray an d gel setup).Pou r 250 ml of refr igerated electrode buffer into eachelectrode tray. Place the tray a nd gel unit in a refr igeratorequipped to circulate cold air . Th e refr igerator shou ld havethe capacity to maintain th e temp erature at 4' C through-out the run.

Enzyme separation is achieved by applying direct cur-rent to the buffered gel. Ions f rom the buffer in the catho daltray en ter the gel. In the A an d B gel systems (see app. B) th eions cause a clearing and a slight depression of the gel at thelocation of the fastest migrating ions. This area is the f ront .We allow the fron t to mig rate 8 cm beyond the origin beforeturnin g off the electric current. Gels of the C a n d D systemsd o no t deve lop f ronts ; the runs a re t imed so as to giveconsistent migration distances between gel runs on differ-ent days.

Caution : Power sources for electrophoresis producedirect current at voltages high enough to cause severeinjury. The possibility of electric shock mus t be eliminatedby c onstant awareness of the electrical hazard, clear com -municatio n between workers, and safe work procedures tominimize shock hazards. All electrical equipment shouldbe examined on a regular basis to verify that wires andconnection s are in safe condit ion. Special care should betaken to protect visi tors f rom electr ical hazards. Newemployees should be th orough ly trained in safe procedures.Electr ical current can be supplied to a gel with aHeathkit-Shumberger high-voltage power source (modelsIP-17 and SP-2717) or similar power sources. The powersources produce 400 volts and 100 milliamps at m aximu msettings. These models, once set, hold constant voltage.Adjus t the amperage m anual ly to the des i red ou tput unt i lthe voltage rises to 320 volts. Power adjustment is notrequired afte r the voltage reaches 320.Each gel is at tached to a pow er source; the current o neach gel is mon itored and adjusted separately. Mo nitor ingeach gel al lows detection of problems such as poo r spongecontact or sep aration of the gels at the or igin; these condi-tions may lead to irregularities in enzyme m igration.To begin the run, connect wires f rom the pow er sourcesto th e electrode terminals of the b uffer trays in the refriger-ator : the cathode (color-coded black) connects to the elec-trode on the or igin side of the gel, the anod e (color-codedred) connects to the opposite electrode. App ly current atam pera ges specified f or the individu al gel buffer systems asfollows:

A buffer system 75 mAB buffer system 70 mAC buffer system 60 mAD buffer system 60 mA

Periodically check the amperag e and voltage gauges on thepower sources and adjust the amperages to maintain therecommended values. Am perages tend to decrease during


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the run . A djus tments that re turn amperages to the recom-mended levels cause an increa se in voltage.Gain experience with the normal range of voltages fordifferent buffer systems, gel thicknesses, and laboratoryconditions. Abnormal voltages may signal problems suchas poo r con tact between the electrode sponges and th e gelor the use of the wrong solution a s tray buffer.

After the current has been o n for 15 minutes , turn off thepower, d isconnect a gel unit, remov e it from the refrigera-tor , remove the clips holding the plastic wrap over thecathodal sponge, and fold back the plastic wrap and thesponge to expose the origin. Use slight pressure with thefingers of one hand to sep arate the gel s lices at the origin.Lift the wicks out of the gel with tweezers. Wh en all wicksare removed from a gel, rejoin the gel slices an d verify tha tthere is good c ontac t between the surfaces. Eliminate bub -bles between the gel and the glass plate. Reassem ble the gelunit with the plastic wrap and the catho dal sponge in theiroriginal positions. Return the unit t o the refrigerator a ndreconnect the electrical wires.Place refrigerated waterbags on the dewicked gels.Waterbags are 20.3 x 30.5 cm heat sealable pouches ofplastic 4.5 mils thick (K apa k/ Sco tchpa k, stock no. 504).Replace waterbags on the gels with a second set of coldbags if there is any tendency for the gels to warm .Reapply the electr ical current at the recommendedamperages for each gel system. Adjust the amperagesthroughout the run until voltages reach 320. Thereafter ,further adjustm ents are unnecessary (amperages will con-tinue to decrease). A sudden dro p in amperage s ignals poorcurrent f low and may be caused by poor c ontact betweenthe electrode sponge and the gel, or separation of the cut gelsurfaces at the origin. Correct poor sponge contact byresetting the sponge. Gel separation a t the origin may bedue t o excessive tension on the covering plastic wrap orunusual heating and cooling conditions within the gel.Correct gel separations by pressing the surfaces backtogether and resetting the plastic wrap and sponges.

When the fro nts on gels of the A a nd B buffer systemsreach 8 cm beyond the origin, disconnect the current. T heC and D system gels do not develop visible fronts and wehave s tandardized the length of t ime for the C and D gelruns at 4.5 hours .

Many stain solutions are perishable an d should be pre-pared as close as possible to the time the gels are ready.

Some chem icals used in s tains are hazardous; several arecarcinogenic. Handle all chemicals with extreme care andan awareness of OSHA guidelines for laboratory safety(U.S . Dep. L abor 1976). Follow the label precautions o n allchemicals.

Remove a gel setup from the refrigerator, unclip theplastic wrap , and fold the electrode sponges into the elec-trode trays. Remove the plastic wrap and the gel moldforms. Enzymes will be in the anodal section of the gelbetween the origin and th e 8-cm fron t. The gel beyond th eanod al s ide of the 8-cm fro nt will not co ntain enzymes andcan be tr immed aw ay and discarded. The catho dal sectionshould be s liced and s tained to locate all bands. C ut onenotch in the upper left hand co rner t o identify gel 1 and twonotches to identify gel 2.Atta ch plastic s tr ips (200 x 25 x 1.0 mm) t o the glaQsoneither side of the gel slab using large spring-loaded clips(fig. 8). Wrap m onofilament nylon sewing thread aro undthe index fingers on both hands and use thumbnails tostretch the line taut. A djust the length of the thread so thatthumbnails r ide on top of the plastic s trips as the cut ismade. S tart with the sewing thread on the far s ide of the geland pull the thread toward you, cutting one s lice throughthe gel. Add a plastic s tr ip at both s ides of the gel and cutthe ne xt slice. Use a new section of sewing thread fo r eachcut. Continue adding spacer strips to slice the entire slab.D o no t sepa rate slices at this time; retu rn the sliced gel slabto th e refrigerator. Trim a nd slice the second gel of the sam ebuffer system.

Consul t appendix C for stain recipes. Stain buffer solu-t ions and some s ta in components can be prepared in quan-tity for use over a period of time. T he stain solutions (withsome exceptions) are mad e up in 75-ml volumes for one-time use. Stain trays a re glass bakin g dishes 20.5 cm squa re(2 qt Pyrex) th at ac com mod ate two gel slices, one fromeach of the two gels of a buffer system. P rew arm t he stainsolutions and staining trays to 37' C to maximize stainactivity w hen the gel slices ar e placed into the solutions.For each enzyme t o be identified, w rite th e abbreviationfo r i t and the da te of the run o n a 5-cm str ip of maskingtape, an d attac h it to a 250-ml Erlenmeyer f lask. Measureout an d add the s tain buffer to the f lask, then ad d and mixthe s tain compone nts in the same order they ar e given in therecipe (ap p. C).Each ba tch of stai n is enough for a pair ofgels.Set out w arm trays and place a f lask of s tain in each tray.Transfer the masking tape from the f lask on to a s tain trayand pour the s tain solution into the tray. Prepare all thestain trays for one gel system.


9/23

Rem ove the sliced gels from the refrigerator; peel off an ddiscard the top slice of the cut gel slab. Use fingertips topeel off the seco nd slice and place it in the first stain trayroceed to p ut one sl ice f rom the f irst gel into eachepeat with slices from the second gel.to cover the gel slices with the stainch tray-with plastic w rap an d follow theincubation recomm endations fo r specif ic stains.ovens will maintain stains at 37' C and shield light-sensitive stains. If ovens are not available, shield light-sensitive stains with a cover of alu mi nu m foil.gels frequently to follow the developm ent ofthe bands on both gels in a stain tray are well resolved,draw off the stain solution and ad d just enough ta p water tocover the gels. The resolution of band s is the clearest at thisstage and data should be collected while the gels are inwater.

To store the gels for 6 to 12 mon ths, replace the water in atray with a fixing solutio n consisting of a 5:5: 1 mixture ofmethano1:water:glacial acetic acid. If the gels ar e not exa m-ined immediately af ter staining, skip the tap w ater step andreplace the stain so lution with f ixing solution. The f ixingsolution tough ens and shr inks the gels and the gels becomeopaque. Dark bands are dist inct af ter f ixing but faintbands may have to be viewed over a light table. Caution:Keep stain trays with fixing solution covered with plasticwrap. Do not inhale the fumes fro m the fixing solution.ethan ol is a poison tha t is only slowly degraded by thebody. Repeated exposure, sometimes over a warm lighttable, may lead t o early signs of poisoning: hea dach e, lossof energy, and fatigue. If possible, work with the fixingsolution only in a vented hood.Gels should remain in the f ixing solution for a minim umplace the gels in fixing solutio n overnigh t,then blot a nd wrap th e f ixed gels in plastic on the followingday. Wra ppe d gels will remain reada ble for long periods oftime if they are protected from desiccation by additionalwraps of plastic and stored in a refrigerator.

en comb ining the starch slurry and boil ing buffer inthe vacuum f lask, wear gloves and safety glasses, and p ointthe vacuum f lask away f rom face and body to avoid thepossible kickback of steam and hot starch.

Never tou ch electrodes or th e tray solutions while cur-rent is supplied to the gels; a severe electric shock ispossible.

om pou nds used fo r staining are hazardousrotective clothing , particularly gloves anda dust mask, when handling. id contaminating otheri tems with these compounds llow prescribed safetyix the stain s in a vented hoo d. Use similar carein examining stained gels treated with fixing solution.

issWork rapidly and keep the sample t issue as cool aspossible to maintain enzym atic activity.Mak e sure that accu rate seed identi ty is m aintained onthe record sheet, in the seed tray, and in the gel.

Degas the hot starch solution no more tha n 20 seconds toavoid congealing of the starch while pouring.Do not al low the gels to f reeze during cooling.

Blot each wick to remove excess moisture.Mak e sure the botto m edge of the wick touches the glassplate.Eliminate bubbles between the gel and the glass at theorigin, after loading an d after dewicking.sisKeep the gels as cool as possible, without freezing;refrigerate and place chilled water bags on th e gels after thedewicking step.

ake sure that the wires to th e electrode trays are no treversed; determine that the p olarity is correct.Hold am perage cons tant at the prescr ibed level unti l agel system reaches 320 volts, then hold the system at 320volts and al low the am perage to decrease for the rest of therun.iciKeep the monofilament thread taut between the thumbs.Be sure to pu t the same nu mb er of plastic spacers on eachside of the gel.Notch the gels for identification.

Warm the staining solutions to 37' C.Because compounds are light-sensitive and perishable,cover trays with aluminum foil and handle chemicals asdirected to preserve their activity.


10/23

Lumps o r white specks appear in hotgel solution

Buffer solution may not have boiledrapidly or the starch may not havebeen well mixed with cold buffer toform the slurry

Discard lum py starch and star t over.Allow buffer to come to a rolling boiland thoroughly mix starch slurry

Starch is too thick or lumpy whenpoured from vacuum flask Starch may have been degassed toolong and is too cool Sta r t over; degas for a shorter t ime an dquickly pour starch into moldVoltage is abnor mally high on a gel Se para tion at origin or poo r spongecontac t with gel After disconnecting power, open plas-tic wrap to inspe ct origin.to improve gel contactFront on gel is irregular: Waterbag unevenly placed

Worn electrode' on anoda l side.(C atho dal electrode is long-lasting)Gel is thicker o n one side than on theo therGel separates in region of origin

Straigh t but higher on one side thanon the other Replace anodal electrode

Level mold before pourin g gel for nextru nPush gel surfaces together at originand place a glass or plastic rod betweengel and b ar o n cathodal end

Wavy Uneven cooling of gel, separation a torigin or po or sponge contact withge l

Check air flow in refrigerator orchange waterbags m ore often. Pushgel surfaces together at origin andplace a glass or plastic rod betweengel and b ar on cathodal end

Gel front migrates slower in one geltha n other gel of same system New sponge may be on cathodal sideof electrode tray Place old sponge in well of cathodetray and new sponge in well of an0d.etrayElectrode may be corrodedUnequal gel thickness

Replace with new electrodeDecrease amperage on gel with fastermigration so that electrophoresis pro-ceeds to same point in same time

Sta in does no t work Possible error in mixing stains orbuffers Check recipe. Som e missed i tems canbe added late and stain may st i ll workPossible deterioration of buffers Ma ke new buffers. Prepare stocks ofbuffers in quantities that will last forabou t 1 monthPossible deterio ration of chemicals Purc hase fresh supplies. Da te eachbottle an d use older supplies first. Fol-low storage instructions on bottles


11/23

ure I-Sample preparation materials. Left: germinated seed inthe petri dish used for germination, and a dissected seed withgametophyte and embryo. Above: grinding block,8.5 X 8.5X 1.3cmclear plastic with 0.7-cm holes inch ) drilled 1.0 cm deep (flatbottom holes were drilled with a bit squared off on a grindingwheel), placed on a frozen pad of Blue Ice for cooli ng (gri ndingblock and pad were frozen together before samples were loadedinto the holes). Below: data sheet with the identity of the samplescoded to the numbered holes in the grinding blocks.


12/23

Figure 2-Gel mold consisting of plastic bars secured with rubberbands to single strength window glass.

Figure 3-Pouring boil ing buffe r into starch suspension.

Gel form

Plastic wrap fold lne-8 m ru n li ne and------

Plastic wrap fold linef:5-> athodal end

'Forms-Glass

4

Side view

ure 4-Degassing hot starch using vacuum line attached to acork in the top of the vacuum flask while blockin g side port of theflask with a finger.


13/23

, elWick spacing guide

section ofgel pulled back toexpose the originGlass base

I Wick spacing guide

Figure 5-Cut gel surface open at the origin. Wicks containing theabsorbed liquid fraction of samples are placed on the vertical sur-face in order corresponding t o their l isting on the data sheet.

Electrode-

Figure&Exact dimensions of electrodetray. The 3/16-inch pure carbon electrodeare secured in to the tray with no. 00 rubbestoppers. The tray is constructed of I/inch lucite.


14/23

ure 7-Side and top views of an electrode tray withgel and wicks, sponges, and plastic wrap in place.

Origin and sponge line Plastic wrap coverPlastic wrap fold line / I 8 cm run line and sponge lineCathodal sponge / I Plastic wrap fold line

Tray buffer

Gel form' \ Electrode tray baseGlass pane

Figure 8-Gel is prepared for slicing by using plastic spacers attached to theglass on either side of the gel slab with spring loaded clips. Above: tautmonofilament n ylon sewing thread cuts horizontal slices through the gel slab.Right: gel slice is placed i n an appropriate stain solution.


15/23

Use pH 7.5, 0.2 phosphate (app. C, Stain bufferformulat ions) as the extract ion buffer. Certain comp ound sadde d to the extrac tion buffer can improve poorly resolvedsets of enzyme bands, and in the initial study of a newspecies , various such compounds should be tested. Ourgeneral rule is to keep the buffer a s simple as possible byincluding only additives that impro ve resolution.Th e add ition of a small qua ntity (20 mg/ 100 ml, buffer)of substrate to the extract ion buffer may improve the reso-lution of specific enzymes. Several. substra tes can be a dde dto the same extract ion buffer (L-glutamic acid for GD H ,D-glucose-6-phosphate for G 6 P D , a-D-glucose I-phos-phate for P GM , and others). From o ur experience, adding

substrates to the extract ion buffer is bet ter than addingthem to the gel or the cathodal electrode buffer as sug-gested in some literature. The substrate in the extractionbuffer prob ably protects the active site of the enzym e whileit is in the mixtu re of cell com pone nts. A fter a sho rt periodof electrophoresis, an enzym e is likely to be isolated fro mcompou nds that degrade i t.Th e resolution of some enzymes is greatly improved bythe additio n of bovine a lbum in (40 mg/ 100 ml) to theextract ion buffer. The a lbum in binds phenolics and freefatty acids (Anderson 1968).Seed resins and phenols can decrease resolution. Theaddit ion of a small quant i ty of 2-mercaptoethanol(1 d ro p/100 ml extract ion buffer) helps to bind and reduce theeffect of resins and phenols. Mercaptoethanol improvesresolution in true firs, incense-cedar, a nd redwood', but wedo not add mercaptoethanol to the extract ion buffer forpines, cypresses, and Douglas-fir.Complex extract ion buffers ar e avai lable for difficul ttissues (Kelley and Adam s 1977, Mitto n an d others 1979,Soltis and o thers 1980); but m ost conifer seed analyses donot req uire these complex buffers.


16/23

. . . . . . . . . . . . . . . . . . . . . . .ormulation Trizma base 62.0 g Lithium hydroxide . . . . . . . . . . . . . . . . . 12.0 gCitric acid . . . . . . . . . . . . . . . . . . . . . . . .14.6 g Boric acid . . . . . . . . . . . . . . . . . . . . . . . . 118.9 gDistilled w ater . . . . . . . . . . . . . . . . . . . . . 0.0 liters Distilled wa ter . . . . . . . . . . . . . . . . . . . . . 10.0 litersDissolve the chemicals an d check the p H. Store at Dissolve the chemicals and check the pH . Sto re atroom temperature room temperature

Procedure To use, ad d 75 ml of the lithium bo rate tray buffer Use as isto 675 ml of the tris citrate gel buffer to m ake therequired 750 mi

Formulation Trizma base . . . . . . . . . . . . . . . . . . . . . . 12 1.1 g Sodium hydroxide ................. 0.0 gDistilled water . . . . . . . . . . . . . . . . . . . . .10.0 liters Boric acid . . . . . . . . . . . . . . . . . . . . . . . . 185.5 gDistilled water. . . . . . . . . . . . . . . . . . . . . 10.0 litersDissolve the trizma base an d titrate to p H 8.8 with Dissolve the chemicals and t i trate t o p H 8.0 with0.2 M citr ic acid solution. Store at room tem- 4N NaOH . Store a t room tempera tureperature

Proce dure Use as is Use as is

Formu lation Trizma base . . . . . . . . . . . . . . . . . . . . . . 162.0 g Sam e as gel bufferCitric acid . . . . . . . . . . . . . . . . . . . . . . . 108.9 gDistilled water . . . . . . . . . . . . . . . . . . . . . . 3.0 litersD i s s o l v e c h e m i c a l s a n d t i t r a t e t o p H 6 . 2with 4N N aOH . R efr igerate

Proce dure To use, mix 16 ml of the buffer with 734 ml of Mix 250 ml of the buffer w ith 750 ml distilled waterdistilled water

Same as gel bufferFormu lation Citric acid . . . . . . . . . . . . . . . . . . . . . . . . 15.4 g Sam e as gel bufferDistilled water. . . . . . . . . . . . . . . . . . . . . . 2.0 liters

Dissolve citric acid an d titrate t o p H 6.1 with N-(3-aminop ropyl)morpho line (20 ml). Refr igerateProcedure To use, mix 37.5 ml of buffer with 712.5 ml of Use full strengthdistilled waterlScandalious 19692Fowler and Morris 19773Nichols and Ruddle 19734Clayton and Tretiak 1972, Yeh and O'M alley 1980


17/23

Enzyme stai ns used on gel slices fro m specific gel systemsare listed in table I. Stock solutions f or chemicals requirein several stain recipes are listed in table 2. Stain buffersolutions ar e listed in table 3. Recipes for preparing stainsare described in table 4. The stains listed in table #a re usedin the analysis of conifer seeds. Additional stains can befound elsewhere in the literature (Harris and Hopkinsonalley and others 1980).Prepare stock solutions for stains in quanti t ies that willbe used up in 4 weeks. Several of the comp one nts of thesesolutions are light-sensitive and perishable. Store themrefrigerated (do not freeze) in dark containers; foil-wraplight-colored containers. Add these components to th estain solutio ns just before add ing the gel slices.

Table I -4tain.s by gel bujyer systemGe1system

StainAbbreviation, Name EC referenceADHAA PESTESTFLESTLAPMNRPEPPERPGMPGIACPCATG6PDGD HGO TSODACONALDIDHSMDHMNRPG DSKDH

Alcohol dehydrogenaseAlanine aminopeptidaseAlpha esteraseBeta esteraseFluorescent esteraseLeucine aminopeptidaseMenadione reductasePeptidasePeroxidasePhosphoglucomutasePhosphoglucose isomeraselAcid phosphataseCatalaseGlucose-6-phosphate

dehydrogenaseGlutamate dehydrogenaseGlutamate-oxaloacetate

transaminase*Superoxide dismutase3AconitaseAldolaseIsocitric dehydrogenaseMalic dehyrogenaseMenadione reductasePhosphogluconate

dehydrogenase6Shikimate dehydrogenase

'Also called glucose phosphate isomerase (GPI) in the literature.*Also called as partate aminotransferase (AAT) in the literature.3Also called tetrazolium oxidase (TO) in the literature.4All of the stains listed for the C system resolve well with the D buffer

system, but bo th systems are necessary to detect var iants within differentloci.

SAlso abbreviated I CD in the literature.hAlso called 6-phosphogluconic dehydrogenase (6PGD) in the litera-

ture.

Prepare stain buffers in quantitie s sufficient to last up to2 months by anticipating laboratory schedules. Thesebuffers ca n be stored at room temperature.

Table 2-Stock solutions for stain components

Table 3-Stain buffer formulationsBuffer

StandardconcentrationAbbreviation

ACP acetate buffer

Name

Aminopeptidasebuffer

G6PDWNADNADPNBTPM S

Catalase buffer

Glucose-6-phosphate dehydrogenase' 5 units/ mlbuffer0-Nicotinamide adenine dinucleotide 10 mg/ ml waterP-NAD phosphate 10 mg/ml wateNitro blue tetrazo lium 10 mg/ ml watePhenazine methosu lfate 5 mg/ ml wate

Esterase buffer

IG6P DH is subject to sulfate ion inhibition. This enzyme is supplied in aconcentrated so lution , which should be diluted using 1 percent bovinealbumin in 0.05 M phosphate buffer (pH 7.5). The albumin binds sulfateions to enhance the activity of the enzyme.

Peroxidase buffer

0.2 M phosphatebuffer

1.0 M trishydrochloride1

1.0 M trishydrochloridei

PH I Formulation. . . . .odium acetate, trihydrate 2.43 g. . . . . . . . . . . .cetic acid , glacial 4.7 ml. . . . ..0 M magnesium chloride 5.0 ml. . . . . . . . . . . . . . .istilled wate r. 1.0 lite

. . . . . . . . . . . . . . . . .rizma base 12.1 g. . . . . . . . . . . . .aleic anhydride 9.8 g. . . . . . . . . . . .odium hydroxide 1.6 g..............istilled wat er. . 1.0 liteSodium phosphate,

. . . . . . . . . . . . . . .onobasic 18.5 gSodium phosphate, dibasic . . . . 17.9 g. . . . . . . . . . . . . . .istilled wate r. 1.0 liteSodium phosphate,. . . . . . . . . . . . . . . .onobasic 13.9 g. . . . .odium phosphate, dibasic 5.3 g. . . . . . . . . . . . . . .istilled water. 1.0 lite

. . . . . .rsenic acid, sodium salt. 5.74 g. . . . . . . . . . . .cetic acid, glacial 1.2 ml. . . . . . . . . . . . . . .istilled wate r. 1.0 liteSodium phosphate,. . . . . . . . . . . . . . . . .onobasic 3.84 gSodium phosphate, dibasic . . . . 23.86 g. . . . . . . . . . . . . . .istilled wate r. 1.0 lite

. . . . . . . . . . . . . . . . .rizma base 74.0 gTrizma hydrochloride. . . . . . . . . 61.4 g. . . . . . . . . . . . . . .istilled wate r. 1.0 lite

. . . . . . . . . . . . . . . . .rizma base 16.0 gTrizma hydrochloride. . . . . . . . 137.4 g. . . . . . . . . . . . . . .istilled water. 1.0 lite

'Dilute these solutions to the concentrations specified in the staicharts.


18/23

Table 4-Stain recipes

EnzymeA C O NAconitasel

Ge lbuffer

75 ml0.2 M tr is HCIpH 8 .0

Cis-aconitic a cid . . . . . . . . . . . . . . . . . . . . . . 150 mgN A D P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . m lN B T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . m l1% MgCl, solut ion . . . . . . . . . . . . . . . . . . . . . . I mlP M S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5 mlIsoci t rate dehydrogenase . . . . . . . . . . . . . . . . 20 uni ts

(0.3 ml)

Stain bufferAdd s t a in comp onen t s t o war m ( 37 ' C) s tain buffer ;incubate gels at 37' C in the dark

A C PAcid phos-phatase?A D HAlcohol de-hydrogenase:

S ta in componen t s

80 mlA C P a c e t a te

buffer

P r ocedur e

a- naph thy l ac id phos pha te . . . . . . . . . . . . . . . 00 mgFas t Garnet GB C Salt ' . . . . . . . . . . . . . . . . . l o 0 m g Allow s tain compone nts t o mix wel l in s tain buffer.Deve lop a t r oo m t emper a tu r e . I nc lude ca thod a l s li ce

of gel in the stain.75 ml0.05 M tris HC1pH 8 . 0

N A D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . mlN B T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . rnlP M S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5 ml. . . . . . . . . . . . . . . . . . . . . . .5% ethyl alcohol . I ml

Add com ponents to warm bu ffer and incubate gels inthe dark

A A PAlanineaminopep-tidase4A L DAld olasel

75 mlAminopep t idas e

bufferL-alanine P-naphthylamide3. . . . . . . . . . . . . . 3 0 m g

dissolved in dimethylsulfoxide . . . . . . . . . . . 2 mlFas t Black K Sal t . . . . . . . . . . . . . . . . . . . . . . . 20 mg

Add com ponents to warm bu ffer and incubate gels inthe dark

75 ml0.05 M tr is HC1p H 8 .0

D-fructose-l,6-diphosphate . . . . . . . . . . . . 250 mgArsenic acid, sodium sal t . . . . . . . . . . . . . . . . 7 5 m gN A D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . mlN B T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . m lP M S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mlGlyceraldehyde-3-phosphate

dehydr ogenas e . . . . . . . . . . . . . . . . . . . . . . 300 uni ts(0.25 ml)

Add components to warm buffer and incubate gels

C AT 100 mlCatalase buffer

. . . . . . . . . . . .% potass ium iodide solut ion 100 ml0.03% H 2 0 2 olut ion, (3% H,O,,. . . . . . . . . . .ml/ 100 ml distilled wate r) ,100 ml

Refr igerate gels in catalase buffer for 30 min. Drainoff buffer and soak in 2% K I for 2 min. Drain off KIand was h twice wi th t ap wate r. Add H 20 2 o lu t ionand score when resolved

E S TAlphaesterase2

75 mlEsterase buffer

Fast Blue R R Sal t . . . . . . . . . . . . . . . . . . . . . . 80 mg1% a-naphthyl acetate so lut ion (dissolve I g

in 50 ml acetone, ad d 5 0 ml dist i lled w ater ,s tore ref r igerated) . . . . . . . . . . . . . . . . . . . . . . ml

Add com ponen t s t o war m buf fe r . Add the naph thy lacetate solut ion late in mixing. Incubate the gels .Include the cathodal slice of gel

E S TBetaesterase

75 mlEsterase buffer

Fas t Garnet GBC Sal t3 . . . . . . . . . . . . . . . . . l o 0 m g1% 2-naphthyl acetate solution (dissolve 1 g

in 50 ml ace tone, add 50 mi dis t il ledwater, store refrigerated) . . . . . . . . . . . . . . . . ml

Add componen t s t o war m buf fe r ; add naph thy l ace-tate to solut ion us t before addinggels . Incubate gels

F L E S TFluorescentesterases

10 mlPeroxidase buffer

4-methylumbel l i feryl acetate solut ion (1 mgdissolved in 3 ml acetone). . . . . . . . . . . . . . . 3 ml Add solut ion t o buffer and paint gels . Score bandson gels within 5 minutes unde r longwave U V light

G D H I S O DGlu tamatedehyd roge-nase, super-ox idedismutaseb

75 ml0.10 M tris HClp H 8 .0

. . . . . . . . . . . . . . . . . . . . . . . . .-glutamic acid - 2 8N A D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . mlN B T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . l m lP M S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 . 5 m l

Add components to warm buffer . Incubate in thedar k . ( S OD bands a r e whi t e aga ins t t he b lue back-ground. )

G O TGlu tamate - 25 ml0.2 M phosphate

bufferpH 7 .5

0.5% pyr idoxal 5-phosphate solut ion(0.5 g/ 100 ml of distilled wa ter). . . . . . . . . 0.8 ml

3.0% bovine albumin solut ion ( 3 g/ 100 mlof distilled water). . . . . . . . . . . . . . . . . . . . . 1.6 ml

0.2 M L-aspar t ic acid (adjus ted to pH 7.5wi th 2N KOH) ....................... .8 ml

Combine f i rs t four components with buffer . Whenready for s taining add the Fas t Blue BB solut ion tocompo nen t s o lu t ion . Add ge l s and deve lop a t r o omtemperatu re. Include a c athodal s l ice of gel

oxaloacetatet ransaminase


19/23

Table 4-Slain recipes (continued)

Procedurenzyme

G 6 P DGlucose-6-phosphatedehyd roge-nasex

ID HIsocitratedehyd roge-naseV

L A PLeucineaminopept i -das e jM D HMalicdehyd roge-nasey

M N RMenadionered uctaseP E PPeptidaseV

?iifer I Stain buffer . . . . . . . . . . . . . . . . .-ketog lutarate solution 2.0 ml.....................ast Blue BB Salt . I 2 0 m g..............issolved in distilled water . 8 m lStain components

Add components to the warm buffer and incubategels in the da rk

. . . . . . . . . . . . . . . . . .-glucose-6-phosphate .20 mg. . . . . . . . . . . . ..0% bovine albumin solut ion . I ml. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A D P l m lNB T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ml1 %M gC 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I ml. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .M S 0 . 5 ml

75 ml0.05 M tris HCIpH 7.0

Add com ponen ts to warm buffer and incubate gels inthe dar k75 ml0.05 M tris HCI

pH 8.0. . . . . . . . . . . . . . . . . . . . . . .L isocitric acid .60 mg

N A D P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ml. . . . . . . . . . . . . . . . . . . . . . . . . . ..25 M MnCl, I mlNB T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ml. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .MS 0.5 ml

Add com ponen ts to warm buffer and incubate gels.4% L-leucine p-napthylamide solution. . . . . . . . .400 mg/ 100 ml distilled w ater ). .5 ml. . . . . . . . . . . . . . . . . . . . . . . . .lack K S a l t . . . 2 0 m g75 mlAminopept idase

buffer

Add co mpon ents to warm buffer and incubate gels inthe dark

. . . . . . . . . . . . . . . . . . . . . .al ic acid solut ion .5 ml(134.1 g DL-malic acid, 80 g NaOH, 1.0liter H,O, adjust to pH 7.0 with about 18ml of 4 N NaOH)

N A D . . . , . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . l m lN BT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ml. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .MS 0.5 ml

75 ml0.05 M tris HC1pH 8.0

Add com ponen ts to warm buffer and incubate gels inthe darkN A D H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 5 mgMenadione . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 m g

N BT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ml0.05 M tris HCIpH 7.0

Com bine f i rs t f ive compone nts with buffer and mixwell. When ready t o stain, add amino-ethyl-carbazolesolut ion and incubate gels

. . . . . . . . . . . . . . . . . . . . . . . .lycyl-L-leucine .I 0 mg. . . . . . . . . . . . . . . . . . . . .-leucyl-L-tyrosine .I 0 mg. . . . . . . . . . . . . . . . . . . . . . . .-valyl-L-leucine 10 mg. . . . . . . . . . . . . . . . . . . . . .eroxidase, crude . I 0 mg. . . . . . . . . . . . . . . . . . . . . . . . . .n a k e v e n om . I 0 m g

50 mg of 3-amino-9-ethyl carbaz ole in 5 ml. . . . . . . . . . . . .f N.N-dimethylformamide . 5 ml

.75 ml0.2 M tris HCIpH 8.0

Add co mp one nts to buffer just before staining. Addgels and develop at room temperature. Use anodaland cathodal slices. Note: origin of gel could bemoved 1 cm toward ano de at beginning of the run forthis system

. . . . . . . . . . . . . . . . . . . ..1 M CaCI, solution . 2 ml. . . . . . . . . . . . . . .-amino-9-ethyl carbaz ole .50 mg. . . . .issolved in N,N-dimethylformamide .5 ml. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .% HH,O, . I mlP E RPeroxidasen

75 mlPeroxidase buffer

Add co mpon ents to warm s tain buffer , add gels andincubate in the dark75 ml0.05 M tris HCI

pH 8.0. . . . . . . . . . . . . . . . .-phosphogluconic acid .20 mg. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A D P 1 ml. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B T 1 ml. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .MS 0.5 ml

6 P G D6-phospho-gluconatedehydroge-nase O

Add al l comp onents to warm buffer . Add gels andincubate in the dark75 ml0.05 M tris HCI

pH 8.0.................-fructose-6-phosphate .25 mg. . . . . . . . . . . . . . . . . . . . . . .% MgCI, solution l ml..................................A D P l m l

N BT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ml. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .MS 0.5 ml. . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 P D H . . .20 units

P GPhosphoglucoseisomeraselo

Add all components to warm buffer. Add gels andincubate in the darka-D-glucose 1.6-diphosphate solution. . . . . . . . . .10 mg: 100 ml distilled w ater ). . I mi. . . . . . . . . . . . . . . .-D-glucose I-phosp hate 140 mg

P G MPhosphoglucomutas eJ O

75 ml0.05 M tris HCIpH 8.0


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Table 4-Stain recipes (con tinued )

1% MgCI, solution ....................... mlN A D P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . mlNB T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . mlP M S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5 mlG 6 P D H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 units

.1.Yeh and O 'Malley 1980 SMitton and others 1979 %ha w and Prasad 19702Scandalious 1969 %haw and Koehn 1968 9Nichols and Ruddle 1973

3Carcinogenic, han dle with care 'Brewba ker and other s 1968 'OBrewer 197040tt and Scandalious 1978'

Stain componentstain buffernzyme

S K D HShikimatedehydroge-nase*

Chemical

ProcedureGelbuffer

. . . . . . . . . . . . . . . . . . . . .,D 75 ml Shikimic acid .75 to 50 mg Add ail com pon ents to warm buffer and incubate in0 .05 M t ri s HCl NA DP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I ml the dark. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .H 8.0 NBT I ml. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .MS 0.5 ml

Catalognumber2

Acetic acid, glacial'A725 1 Cis-aconitic a cidA2628 L-alanine P-napthylamide3A4503 Albumin, bovine, fraction V powderA5754 3-amino-9-ethylcarbazole12,390-94 N-(3-aminopropyl)morpholine3A6756 Arsenic acid, sodiu m salt3A9256 L-aspartic acid, free acidB0252 Boric acidC0759 Citric acid, free acid, anhydro usD4254 N,N-dimethylformamide3D5879 Dimethyl sulfoxide

Ethanol, 95%F7253 Fast Black K SaltF3378 Fast Blue BB Salt3F0500 Fast Blue RR Salt3F0875 Fast Garnet GBC Salt3752-1 D-fructose 1,6-diphosp hate, trisod ium saltF3627 D-fructose-6-phosphate, sodium salt , grade 1G5875 a-D-gluco se 1,6-diphosp hate, tet ra(cyclohexylammonium)

saltG7000 a-D glucose I-phosph ate, disodium salt: tetrahydrateG7879 D-glucose-6-phosphate, monos odium saltG8878 Glucose-6-phosphate dehydrogenase, Type XIG 1626 L-glutamic acid, monoso dium saltG5 126 Glyceraldehyde-3-phosphate dehydrogenaseG2002 Glycyl-L-leucine

Hydrogen peroxide (3%)I1252 DL-isocitric acid, trisodium salt12002 Isocitric dehy drog enas e, Type IV410-2 a-ketoglutarate solution, 0.1 M, pH 7.5 in 0.1 M phosphatebufferLO376 L-leucine P-na phthy lamid e, hydroch loride3LO501 L-leucyl-L-tyrosineL4256 Lithium hydroxide, monohydrate3

Magnesium chloride, hexahydrateMaleic anhydride3DL-malic acidMenadione2-mercaptoethanolMethan ol, 95%34-methylumbelliferyl acetatecx-naphthyl acetate32-naphthyl acetate3a-naphthyl acid phosphate, monosodium salt3P-nicotinamide adenine dinucleotide (NA D)P-nicotinamide a denine dinucleotide, reduced form(NADH), disodium saltP-nicotinamide adenine dinucleotide phosphate (NADP),

sodium saltNitro blue tetrazolium (NBT)3Peroxidase, type 11Ph'enazine methosulfate (PMS)36-phosphogluconic acid, trisodium saltPotassium hydroxide3Potassium iodidePyridoxal 5-phosphate(-)shikimic acidSodium acetate, trihydrateSodium hydroxide, anhydrous pellets3Sodium phosphate, monobasicSodium phosphate, dibasicStarc h, hydrolyzed for electrophoresisTrizma baseTrizma hydrochlorideL-valyl-L-leucineVenom, snake (Crotalus atrox )

'M any chemicals are available in different forms, with different saltsand hydration levels. Sigma and Aldrich Chemical Company catalognumbers a nd chemical names are supplied to avoid confusion for all butthe most comm on chemicals-glacial acetic acid, ethanol, hydrogenperoxide, and methanol.

2Sigma Chem ical Co., P.O. Box 14508, St. Louis, M o. 63178.3Hazardous chemical, handle with care.4Aldrich Chemical Co., 940 West Saint Paul Avenue, Milwaukee, Wis.53233.5Additional brand s of starch are Fisher, Connaug ht, and Eiectro-starch.


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lant tissues. Phytochem istry 7: 1973-1988; 1968.

te PS W-264. Berkeley,riment Station, Forest

Springer-Verlag; 1976; 49-77.Fowler, D. P.; Mo rris, R. W. Genetic diversity in re pine: evidence for lowgenetic heterozygosity. Can. J.

H arry; Hopkinson, D. A.genetics [with supplemCo.; New York: Oxford American Elsevier Publishing Co.;

1976.Kelley, Walter A.; A dams, R obert P. reparation of extracts frooresis. Phytochem istry I6:5 13-5 16; 1977.

21:1066-1081; 1973.O'Malley, David; Wheeler, Nicholas C ;Guries, Raymond P. Aoresis. Staff Paper Series 11. Madison, W1:Department of Forestry, College of Agricultural and Life Sciences,

,Gerald J. Detect-i ~ : n analysis of

ment of Agriculture; 1974. 883 p.

Genet. 2993-92; 1980.


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, s responsible for Federal leadership inforestry. It carries out this role through four main activities:@ Protection and management of resources on 191million acres of National Forest System lands.@ Cooperation with State and local governments, forest industries, and private landowners to

help protect and manage non-Federal forest and associated range and watershed lands.e Participation with other agencies in human resource and community assistance programs to

improve living conditions in rural areas.eland management, and forest resources utilization.

a, Hawaii, and the westernPacific.


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Conkle, M. Thompson; Hodgskiss, Paul D.; Nunnally, Lucy B.; Hunter, Serena C.PSW-64. Berkeley, CA : Pacific Southwest Forest and R ange Experiment Stat ion,Forest Service, U.S. De par tmen t of Agriculture; 1982. 18 p.This m anu al describes fast, low-cost biochem ical procedures for separa ting enzymes

representing numerous genes of forest trees. During electrophoresis the mixture ofenzymes from a meg agam etophy te or embryo of a germinated seed separates in a gel.Specific stains applied to gel slices locate each enzyme. These procedures expan d onthose developed for crops research. They provide a means for forest genet icists to geturgently needed information on the amount and geographic distribution of geneticvariation in conifers for evaluating species relationships, for protecting rare naturalpopula tions, an d for deciding on breeding progra ms fo r commercial species. Electro-phoresis of conifers is an altern ative to long-term, high-cost field trials.Retrieval Terms: technique, gam etophyte s, embryos, allozymes, eiectrophoresis

starch gel electrophoresis of conifer seeds: a laboratory manual

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