lab 4 protein purification by ion exchange student...

BIOTECHNOLOGY I – PROTEIN PURIFICATION BY ION EXCHANGE

Written by Eilene Lyons Revised 2/25/10 4-1

LAB 4

PROTEIN PURIFICATION BY ION EXCHANGE

STUDENT GUIDE

GOAL

The goal of this laboratory is to teach the basics of ion exchange chromatography, DNA

restriction, and specific activity of enzymes.

OBJECTIVES

After this lab, the student will be able to

1. Describe the molecular action of Type II endonucleases.

2. Explain why cellular DNA is not degraded by the cell’s endonucleases.

3. Set up and use a chromatography column to purify a protein from cell extract.

4. Assay fractions from column chromatography of bacterial cells for DNA restriction enzyme

concentration

5. Calculate the units of activity and the specific activity of an enzyme from experimental

results.

6. Describe the difference between total activity and specific activity of an enzyme.

7. Apply knowledge of unit activity and specific activity when ordering enzymes.

TIMELINE

Day 1 – Prep, collect column fractions, cast agarose gel

Day 2 – First Assay

Day 3 – Second Assay and data analysis

BACKGROUND

Restriction Enzymes

Bacteria use restriction enzymes to destroy invading virus DNA by cutting it into fragments along

the sugar-phosphate backbone of the DNA. Immediately after DNA replication, bacteria protect

their own DNA by adding methyl groups to restriction recognition sites recognized by their own

restriction enzymes, preventing binding of these enzymes to the cellular DNA. Some restriction

enzymes cut from a free end of the DNA (exonuclease), while others cut along the backbone

(endonuclease). Since Type II endonucleases cut at sequence-specific sites and are the type of

enzyme used most often in genetic engineering, they are referred to simply as restriction

enzymes in the laboratory. Over 1500 different restriction enzymes have been found since the

first was discovered by H. O. Smith and colleagues in 1968. Most restriction enzymes are either

protein dimmers (two subunits of equal mass between 20,000 and 25,000 daltons) or single

polypeptides of molecular weights from 30,000 to 35,000 daltons. Different restriction enzymes

use different recognition sequences (usually palindromes) of DNA where they attach and cut. A

palindrome is a word or phrase spelled exactly the same forward and backward. DNA

palindromes involve both strands of the molecule, where the sequence on the bottom strand is the

opposite of the complementary base pair sequence on the top. For example,

GAATTC

CTTAAG



Different species have different restriction enzymes, so each has been named according

to the species where they were discovered. The Roman numeral denotes if the enzyme

was the first, second, third, etc., enzyme found in the species. Some restriction enzymes

recognize the same DNA sequence and are referred to as “isoschizomers.” The restriction

enzymes Cla I and Bsp 106 both attach to the DNA sequence ATCGAT and cut between

the T and C. Molecular biology product catalogues give isoschizomer lists and sequence

recognition sites for most enzymes used in the research laboratory. There are reports in

the scientific literature of restriction enzymes cutting at more than one recognition site

due to changes in the cation concentration, the pH of the solution, or the presence of

glycerol in the reaction mixture. This indiscriminate cutting is referred to as star activity,

and can also be caused when the restriction enzyme concentration is too high.

Some endonucleases, like EcoR V, cut leaving blunt ends. Others cut asymmetrically,

leaving cohesive or sticky ends – nucleotides that are not hydrogen bonded to other

complementary nucleotides. Once cut from within, DNA polymers separate as the

hydrogen bonds between the cuts break. See Table 1.

5’ CGAATG AATTCTACCCAAG 3’

3’ GCTTACTTAAG ATGGGTTC 5’

Table 1. Examples of Restriction Endonuclease Recognition And

Cutting Sites ( and spaces denote where the DNA backbone is cut.)

EcoR I: G AATTC G GATCC BamH I:

Escherichia coli, CTTAA G CCTAG G Bacillus amyloliquefaciens H

strain RY 13

Hind III: A AGCTT GAT ATC EcoR V (a blunt cutter):

Haemophilus TTCGA A CTA TAG Escherichia coli,

influenzae Rd strain RY 13

PRACTICE: Identify the restriction site in each DNA sequence below. Write the name of the

enzyme on the line beside each sequence. Mark the cutting sites with slashes. Draw a horizontal

line showing what hydrogen bonds will break, separating the two polymers of DNA.

A. B. TAGACTGAATTCAAGTCA ATACGTAGGATCCCTAAA

ATCTGACTTAAGTTCAGT ________ TATGCATCCTAGGGATTT ________

C. D.

TTACTGATATCCATATGC TCGTTAAGCTTATGCCAT AATGACTATAGGTATACG ________ AGCAATTCGAATACGGTA ________

Sticky

ends



Restriction of Lambda DNA by Eco RI

The most widely used method for determination of a restriction enzyme’s cutting ability

is to assay it using DNA from a small viral genome and running the resulting fragments

on an agarose gel for analysis. To assay for Eco RI activity in this lab, a sample of each

fraction collected by column chromatography will be incubated with chromosomal

Lambda viral DNA to see which of the fractions cuts the DNA. To determine cutting, the

DNA restrictions digestions will be applied to an agarose gel to determine if all the

known Eco RI sites are completely cut, giving an expected number of fragments on the

gel. There are 5 Eco RI sites in the Lambda chromosome, giving linear fragments of the

following sizes when cut: 21226, 7421, 5804, 5643, 4878, 3530 bp. The two fragments of

5804 and 5643 bp are so similar in size that there may not be separation to show two

distinct bands if the gel is not run long enough. Therefore, you may see either 5 or 6

bands on the gel, depending on how long it was electrophoresed and whether there was

enough Eco RI present to cut the DNA completely.

Ion Exchange Chromatography

Purification of restriction enzymes can be accomplished using Ion Exchange

Chromatography. In column chromatography, a resin or column packing compound, such

as polystyrene or a polysaccharide such as Sepharose, Sephadex, or Diethylaminoethyl-

Cellulose (DEAE-Cellulose), is “packed” into a column. The resin must be kept moist

with a buffer that covers it so that it does not dry out, preventing it from functioning

properly. The sample containing the protein of interest is applied to the top of the column

and allowed to enter the column as drops of the buffer are collected off the bottom (called

“fractions”). See Figure 1. The proteins in the sample bind to the charged ions of the

column, and the column is washed with buffer at a pH and salt concentration that

maintains the interaction of the proteins with the resin. The salt concentration of wash

buffer is gradually increased so that only the protein of interest binds tightly. As the salt

concentration is increased further, the protein of interest releases (elutes) from the resin

and will drip through the column. Measured fractions of wash and elution buffer are

collected and then tested for desired enzyme activity.

Figure 1. Steps of Ion Exchange Chromatography

Bound protein of interest

Other proteins



Calculating Total Activity The activity of an enzyme can be measure in two ways, as units of activity or as specific

activity. A Unit of activity is defined as the amount of enzyme that fully cuts a given

amount of substrate in a given amount of time under standard assay conditions. For

example, if the assay conditions are that 1 µg of DNA is used and that the reaction is to

be incubated for 1 hour, resulting in complete cutting of the DNA as determined by gel

electrophoresis, then the enzyme volume used is equivalent to 1 Unit of activity. See the

sample problem, below.

Calculating Specific Activity

Specific activity is the number of Units of activity per milligram of protein present in the

sample. Specific activity is a way of saying how much of the protein in the fraction is the

enzyme of interest, in this case, EcoR I. In industry, specific activity is a measure of

purity - how much enzyme there is per the total protein in the sample. This has a bearing

on how to select enzymes for purchase. You may find several forms of an enzyme from a

chemical supplier, some containing more Units of activity, while others show higher

specific activity. The amount of purity may be more important than the cost or more

important than how much you must use for your application, in which case, you would

opt to pay more and get the more highly purified enzyme rather than the cheaper, less

purified one. To calculate the specific activity of the enzyme from the sample problem,

above, you would first need to take a UV spectrophotometer absorbance reading to get

the milligrams of protein per milliliter. Suppose that the A280 was 0.234. Recall that the

spec reading at 280 nm is the milligrams of protein per milliliter that are present in a

sample. The calculation of specific activity would then be:

Sample Problem: By definition, one Unit of Eco R I enzyme activity is equal to the amount of enzyme that can digest 1 µg of genomic Lambda viral DNA in 60 minutes at 37°C. Units of activity are expressed as Units per milliliter of enzyme solution. A sample of the fraction is diluted 1 to 4. Ten microliters of the dilution showed complete

digestion of 1 g of Lambda DNA in 30 minutes. The amount of enzyme in that 10 µl of fraction (we don’t know its molar amount) is equivalent, therefore, to 1 Unit of activity. Multiply by the dilution factor (4) and by 2, for how much would be cut if you incubated for twice that long (since by definition 1 unit is how much enzyme it takes to cut the DNA in one hour).

(1U/10 l) x 4 x 2 = 8U/10 l = 0.8 U/l

To calculate how much is in 1 ml of the fraction:

(0.8 U/l) x 1000 l/ml = 800 U/ml

Specific Activity = (800 U/ml) ÷ 0.234 mg/ml = 3,418 U/ mg protein



Buying Enzymes for Research

Here are some points to consider before you buy an enzyme:

1. What is the application? Will the protocol make a difference?

2. How pure does the enzyme need to be, i.e., will other contaminants have an effect

on the experiment?

3. What is the size of the reaction for which the enzyme will be used? Is it better to

use a small amount rather than a large amount of enzyme in a single reaction in

order to get the substrate completely degraded?

4. How important is the cost? Is there a minimum amount you can spend or do you

want what is best for the purpose, regardless of the price?

5. How long does the enzyme keep? If it has a long shelf life, it may be better to buy

more so that you pay less per mg.

6. How many units will be needed based on previous tests?

LAB OVERVIEW In this lab, the restriction endonuclease Eco R I will be isolated from a bacterial cell

lysate by ion exchange chromatography. The fractions collected will be assayed by

incubation with Lambda chromosomal DNA. Units of activity and specific activity of the

isolated enzyme will then be calculated.

SAFETY GUIDELINES

Good Laboratory Practice requires wearing safety glasses and gloves. Use appropriate

safety precautions during gel electrophoresis as directed previously.

MATERIALS EQUIPMENT

Per class: Chromatography columns, one per Team

E. coli RY extract, lyophilized Ring stand with clamps, one per Team

DEAE-Cellulose Ten 13 x 100 mm test tubes per Team

10x Equilibration Buffer Gel electrophoresis units, one per Team

50% Glycerol 5 ml serological pipette and pump, one per Team

KCl Power supplies, one per two Teams

Eco RI Reaction Buffer Waterbath set at 37°C (for digestions); 65°C for

electrophoresis and 1.5 ml tube floats

Molecular grade water (Qualified water) UV Spectrophotometers, two

Lambda DNA Matched Quartz Cuvettes & cuvette rack

Lambda/Eco RI Marker Automatic micropipetters and tips

Eco RI Dilution Buffer Two microcentrifuges

10x Gel loading dye Microwave oven

50x TAE Electrophoresis Buffer Balances, spatula, weigh boats, cleaning brush

Agarose powder Ice buckets, ice

Ethidium Bromide [10 mg/ml] UV Transilluminator, camera, film

1 x TNE Buffer for UV analysis Dishes for transporting gels

Plastic wrap and parafilm

Lab diapers for staining station

5 ml serological pipette for rolling out bubbles

Sharpie markers



PROCEDURE

NOTE: The reagents you receive are for two assays. The kit contains enough

reagents for 6 Teams.

Part I. Prep

(Some of these steps may be performed by laboratory personnel.)

DEAE-Cellulose Matrix

Add 35 ml of 10x equilibration buffer (C) to the bottle of DEAE-Cellulose (B). Cap

tightly and place on a rocker or orbital shaker to hydrate for 30-60 minutes. Aliquot 6 ml

for each of the six Teams.

1x Equilibration Buffer

(NOTE: The 10x Equilibration buffer contains potassium phosphate, pH 7.4, EDTA, and

β-mercaptoethanol.) Mix the following and stir thoroughly:

350 ml dH2O

50 ml 10x Equilibration buffer (C)

100 ml 50% glycerol (D)

KCl Buffers

0.1 M KCl: use 0.75 g KCl and BTV of 100 ml with 1x Equilibration Buffer



E. coli Cell Extract containing Eco RI restriction enzyme 1. Re-hydrate the sample by adding 0.5 ml of ddH2O to tube component A and let sit for

5 minutes.

2. Mix by vortexing on high speed and transfer the entire contents to a 50 ml conical

tube. Rinse tube A six times – each time with 1 ml of 1x Equilibration Buffer and add

the rinse material to the 50 ml conical tube. Mix the contents well.

3. Label a tube for each of 6 lab Teams as “Cell extract” and add 1 ml of the re-hydrated

extract to each tube. Store this extract on ice.

Team 1

1. Obtain a test tube rack for each team. Label a 15 ml conical tube for each team as

“0.1 M KCl.” Dispense 6 ml of 0.1 M KCl into each tube and distribute to each

team’s test tube rack. Distribute racks.

2. Label a 1.5 ml tube for each team as “Rxn Buffer” and dispense 100 µl of Eco RI

Reaction Buffer (F) into each tube. Distribute to each team’s ice bucket.

Team 2

1. Set up an ice bucket for each team and turn on water bath – set at 37°C.

2. Label a 50 ml conical tube for each team as “1x Equilibration Buffer.” Dispense 35

ml of 1x Equilibration Buffer into each and distribute to each team’s ice bucket.



Team 3

1. Label a 1.5 ml microfuge tube for each team as “Lambda DNA” and dispense 100 µl

of Lambda DNA (H) into each tube. Distribute to each team’s ice bucket.

2. Label a 15 ml conical tube for each team as “0.2 M KCl.” Dispense 6 ml of 0.2 M

KCl into each tube and distribute to each team’s test tube rack (Team 2 may have the

racks at their bench).

Team 4

1. Label a 1.5 ml microfuge tube for each team as “10x Loading Dye” and dispense

100 µl of 10x Loading Dye into each tube. Distribute to each team.

2. Label a 15 ml conical tube for each team as “0.5 M KCl.” Dispense 6 ml of 0.5 M

KCl into each tube and distribute to each team’s test tube rack.

Team 5 (or Team 3, if there are only 4 Teams)

1. Label a 1.5 ml microfuge tube for each team as “-Eco RI Marker DNA” and

dispense 50 µl of Lambda/Eco RI Marker (I) into each tube. Distribute to each team.

(Note: is lab shorthand for Lambda.)

2. Label a 1.5 ml microfuge tube for each team as “Dilution Buffer” and dispense 250 µl

of Eco RI Dilution Buffer (J) into each tube. Place these tubes in the class freezer

box. They will not be needed until the second assay.

Each team should check to make sure they have the following at their bench:

Ice Bucket: On bench: In rack

*Q-Water 100 ul 10x Loading Dye 6 ml 0.1 M KCL

100 ul Rxn Buffer (F) 50 ul Eco RI Marker DNA 6 ml 0.2 M KCl

100 ul DNA (H)

[0.2 μg/μl]

1 chromatography column 6 ml 0.5 M KCl

1 ml Cell Extract (A) 1 ring stand with clamp 35 ml 1x Equilibration Buffer

250 ul dilution buffer (J) 9 glass test tubes (13 x 100 mm) 6 ml DEAE cellulose matrix

25 ul dilute DNA

[4 ng/μl] not required until first

assay

*Q-water = ddH2O = millipore H2O = molecular grade water

Automatic pipetters and tips

5 ml serological pipettes and pump

A supply of 1.5 ml microcentrifuge tubes

Beaker for used tips

REMEMBER to save all reagents for the next 2 labs when the assays will be conducted.



PART II. Preparation of the Column

Perform the column separation of the Eco RI at room temperature and then place the

fractions on ice or store at -20°C until the next lab session.

Packing and Equilibrating the Column

1. Attach the column to a ring stand, making sure it is vertically straight.

2. Make sure the cap is on the bottom of the column.

3. Mix the DEAE-Cellulose matrix by gentle inversion.

4. Use a serological pipette to gently add the matrix to the column by inserting the

pipette tip down to the bottom and touching the inside of the column. Add the matrix

so as not to trap any air bubbles (see Figure 2, below). If you see an air bubble get

trapped, stop and gently tap or flick the column to dislodge it so that it comes to the

surface and pops. The matrix should be smooth on top when you finish.

Matrix is smooth and has no trapped air bubbles.

Addition of the matrix to the side of the column

Equilibration Buffer DO NOT LET THE COLUMN GO DRY!

FIGURE 2. PREPARATION OF THE COLUMN



5. Place a small beaker underneath the column. This will be used to collect the wash

from the column as you drain it.

6. Remove the cap from the bottom of the column, allowing the DEAE-Cellulose

matrix to drain out, but DO NOT LET THE COLUMN GO DRY!"

7. After the column drains, add 25 ml of 1x equilibration buffer and let it drain again

until the meniscus is just above the surface of the matrix. Replace the end cap to stop

the draining.

8. Replace the cap so that a meniscus of the Equilibration Buffer remains on the top of

the matrix.

PART III. Collecting Fractions

1. Using a Sharpie marker, label eight 13 x 100 mm test tubes numbers 1 – 8. The chart

below indicates what each collected fraction in each tube will contain.

Tube Collected Fraction

1 Equilibration buffer

2 Equilibration buffer

3 0.1 M KCl

4 0.1 M KCl

5 0.2 M KCl

6 0.2 M KCl

7 0.5 M KCl

8 0.5 M KCl

2. Prepare a reference tube by adding 3 ml of water. Use this reference tube to mark the

3 ml point on each of the 8 collection tubes with a Sharpie marker. You will collect

eluent to this line in each of the tubes, one after another.

3. The meniscus should be just at the surface of the matrix. Use a P1000 automatic

micropipetter or a 1 ml serological pipette to slowly add 1 mL of the Eco RI cell

extract to the middle of the surface of the matrix so as not to disturb it. Remove the

cap and drain so that the buffer plus cell extract enters the surface of the matrix. See

photo, below.



4. Just as the surface of the liquid meets the surface of the matrix, slowly add 6 ml of

Equilibration Buffer to the inside of the column using a 5 ml serological pipette and

pump. Place tube number 1 under the column and collect 3 ml of the Equilibration

Buffer eluent. Switch to tube 2 and collect 3 mL more of eluent. Place these

collection tubes on ice and proceed directly to the next step without letting the

column drain out completely.

5. Add 6 mL of 0.1 M KCl to the top of the column and collect 3 mL fractions in tubes

3 and 4. Place on ice.





8. Wrap the top of each tube with Parafilm and place all into a test tube rack for storage

at -20°C until the next class session when the first assay will be performed.

9. Clean up your lab bench, dispose of the column, and proceed to casting your agarose

gel.

Add the cell extract just when the buffer reaches the top of the matrix.



PART IV. Casting an 0.8% Agarose Gel

Prepare an 0.8% agarose gel as done previously. Place it in a labeled zippered bag for

storage at 4°C until the next laboratory session.

PART V. First Assay of Eco RI Activity

Set up two water baths: one at 37°C and one at 65°C.

Note: Be careful not to get the Eco RI dilution buffer and Eco RI reaction buffer

confused. There are three different samples of DNA used in this lab: EcoR I markers,

chromosomal [0.2 ug/ul] Lambda DNA for the assays, and dilute Lambda DNA to run as

an uncut control on the gels.

1. Make sure the water baths are on and set and that your collected fractions from

the last lab are on ice and completely thawed.

2. Label nine 1.5 mL microcentrifuge tubes 1-9, Team number and date. To tube #9,

add the following:

Tube 9 – The Master Mix 270 µl molecular grade water

45 µl Eco RI Reaction Buffer

45 µl Lambda DNA [0.2 ug/ul]

360 µl total volume

3. Cap tube 9, flick to mix, and pulse spin (centrifuge briefly) to move liquid to the

bottom.

4. Transfer 40 µl of this master mix to each tube 1-8, leaving 40 µl in tube 9.

5. To tubes 1 – 8, add 10 µl of the corresponding Eco RI fraction to give a total

volume of 50 µl. Immediately pulse spin to mix and place into the 37°C water

bath for exactly 30 minutes. Tube 9 is the negative control and will have no

chromatography fraction added. Instead, add 10 µl of molecular grade water, cap

it and incubate with the rest of the test samples.

6. DO NOT DISCARD THE 9 TEST TUBES WITH THE CHROMATOGRAPHY

FRACTIONS. They should be returned to -20°C until the second assay during the

next laboratory session.

7. At the end of the 30 minute incubation period, remove the tubes from the 37°C

water bath and add 5 µl of 10x loading dye to all 9 tubes. The loading dye stops

the action of the restriction enzyme.

8. Set up the gel electrophoresis chamber using your 0.8% agarose gel and 1x TAE

Electrophoresis Buffer.



9. Heat the Lambda DNA standard markers and the Eco RI assays at 65°C for two

minutes. Pulse spin to move all liquid to the bottom of each tube. Place on ice and

load the gel in the following order:

Lane 1. 20 µl digestion #1 (Equilibration buffer fraction)

Lane 2. 20 µl digestion #2 (Equilibration buffer fraction)

Lane 3. 20 µl digestion #3 (0.1 M KCl fraction)






Lane 9. 20 µl negative control #9 (no fraction added)

Lane 10. 20 µl DNA Eco RI Markers (+ control)

10. Add 25 μl of [10 mg/ml] Ethidium bromide to the buffer and run the gel at 90-95

volts until the dye has migrated at least 3 cm.

11. Photograph the gel as done previously. Give your gel photo to the instructor for

duplication for your lab partner(s) and for the instructor.

12. Analyze the results to determine which fractions contained enough Eco RI

restriction enzyme to cut the Lambda DNA.

PART VI. Second Assay of Eco RI Activity

Set up two water baths that will hold all group samples in each: one at 37°C and the other

at 65°C. Note: Be careful not to get the Eco RI dilution buffer and Eco RI reaction buffer

confused. There are three different samples of DNA used in this lab: EcoR I markers,

chromosomal [0.2 ug/ul] Lambda DNA for the assays, and dilute Lambda DNA to run as

an uncut control on the gels.

1. Make sure the water baths are on and up to temperature. Thaw your original

column chromatography fractions. Use the results from the first assay to

determine which fractions had cutting activity and pool these fractions. Pool

together only those fractions that gave significant cutting, not the ones that

showed only slight cutting as this will dilute those fractions with better activity.

2. Label two 1.5 µl microcentrifuge tubes as 1/2 dilution and as 1/3 dilution. Use the

Eco RI dilution buffer to mix the dilutions as followings:

1/2 dilution 1/3 dilution

Pooled fractions: 15 µl 10 µl

Eco RI dilution buffer 15 ul 20 ul

Total Volume 30 µl 30 µl



3. Label a fresh 1.5 ml microfuge tube as “Master Mix.” Prepare the master mix of

molecular grade water, Eco RI reaction buffer and Lambda DNA as follows:

“Master Mix” for digestion reactions:

Molecular grade water: 300 µl

Eco RI reaction buffer 50 µl

Lambda DNA [0.2 μg/μl] 50 µl

Total Volume: 400 µl

4. Label ten 1.5 ml microcentrifuge tubes as 1-10 with your Team number and date

on each. Use the following table to add components to each tube. One partner can

set up tubes 1 – 4, the other tubes 6 – 9. It is important to add the Eco R I to each

set of four tubes at about the same time so that some reactions do not have more

time to digest than others. Add master mix and water to tubes 5 and 10 last, just

in case there are slight pipetting errors in master mix aliquoting.

Last!

Digestion Tube: 1 2 3 4 5 6 7 8 9 10

Master Mix 40 ul 40 ul 40 ul 40 ul 40 ul 40 ul 40 ul 40 ul 40 ul 40 ul

Additional MG water ----- ----- ----- ----- 10 µl ----- ----- ----- ----- 10 µl

Eco RI 1/2 dilution 10 ul ----- ----- ----- ----- 10 ul ----- ----- ----- -----

Eco RI 1/3 dilution ----- 10 ul ----- ----- ----- ----- 10 ul ----- ----- -----

Undiluted Eco RI from

pool of fractions

----- ----- 10 ul 20 ul ----- ----- ----- 10 ul 20 ul -----

Total Volume 50 ul 50 ul 50 ul 60 ul 50 ul 50 ul 50 ul 50 ul 60 ul 50 ul

Time of Incubation at 37°C 30

min.

30

min.

30

min.

30

min.

30

min.

60

min.

60

min.

60

min.

60

min.

60

min.

5. Pulse spin all. Place all tubes in a 37°C water bath. Incubate tubes 1 – 5 for

exactly 30 minutes. Incubate tubes 6 – 10 for exactly 60 minutes.

6. While the samples are incubating or while the gel is running, determine the

amount of protein in your pooled fractions. Dilute 1 µl into each ml of 1x TNE

buffer in a quartz cuvette and take a UV spec reading at A280. A blank matching

quartz cuvette with 1x TNE buffer must be used. Take three readings of your

pooled sample and average the absorbance readings. An absorbance reading of 1

unit contains 1 mg of protein. Calculate the amount of protein per milliliter.

Record your readings and calculation in your lab notebook.

7. At the end of the incubation period, remove the tubes from the 37°C water bath

and add 5 µl of sample loading dye to each tube to stop the reaction.

8. Heat the Eco RI assays at 65°C for two minutes. Pulse spin to move all liquid to

the bottom of each tube. Place on ice until ready to perform gel electrophoresis

9. After all incubations are finished, heat the Lambda DNA markers and the Eco RI

assays at 65°C for two minutes. Pulse spin to move all liquid to the bottom of

each tube. Place on ice.

Last!



10. Insert the E-gel into the holder and run for 2 minutes with the comb still in the

wells. (NOTES: A steady, red light illuminates on the base if the gel is correctly

inserted. Press and hold either button until the red light turns to a flashing-green

light, indicating the start of the 2 minute pre-run. It will shut off automatically

after 2 minutes).

11. Remove the comb and load the samples in the following order:

Lane 1 20 µl Tube 1 – 1/2 dilution pooled fractions (30 min)


Lane 3 20 µl Tube 3 – 10 µl undiluted pooled fractions (30 min)


Lane 5 20 µl Tube 5 – no Eco RI fraction (- control) (30 min)


Lanes 7 20 µl Tube 7 – 1/3 dilution pooled fractions (60 min)

Lanes 8 20 µl Tube 8 – 10 µl undiluted pooled fractions (60 min)


Lane 10 20 µl Tube 10 – no Eco RI fraction (- control) (60 min)

Lane 11 20 µl Lambda – Eco RI MWt marker DNA (+ control) (60 min)

Lane 12 20 µl uncut Lambda DNA [4 ng/μL]

12. Run the gel for 30 minutes, view on the transilluminator, and document for your

lab notebook.

DATA ANALYSIS

Determine the units of activity per milliliter and the specific activity of the EcoR I

enzyme that you purified from the cell extract. The Lambda DNA added to the master

mix had a concentration of 0.2 μg/μl and 50 μl was added. Therefore, the total amount of

Lambda DNA in the master mix was 0.2 x 50 = 10 μg. Into each of the 10 assay tubes

was added 40 μl for a total of one microgram of Lambda DNA. This makes calculation of

the units of activity much easier.

10μg/400 μl = 1 μg/40 μl

In the second assay, the pooled fraction combo from the first assay was diluted to observe

the smallest dilution that gives complete cutting of 1 g of DNA during incubation. The

dilution you should use in your calculation for Specific Activity is the one that gave

complete cutting in the least amount of time with the least volume of pooled fraction

used.



QUESTIONS

1. If you do not see complete digestion in any of the second assays, can you

accurately calculate the total activity present? (HINT: Would you know how

much of the combined fraction is required to cut 1 g of the DNA?)

2. Explain how ion exchange chromatography works to isolate a particular cellular

product. Use lecture notes, books on reserve, Internet sources, etc., but document

the information with appropriate citations.

3. How is E. coli DNA protected from digestion by Eco RI while within the living

cell?

4. How many Eco RI sites are there in the Lambda genome? How many bands were

evident on the gel electrophoresis results? Explain any discrepancy.

5. Explain the difference between units of activity and specific activity in a sample.

6. If the specific activity of a restriction enzyme is 5U/mg, how much would be

required if you wanted to digest 1 μg of DNA? Show your work or explain your

reasoning.

7. Your lab is studying how to genetically engineer potatoes so that they contain

25% more starch and less water. This would be a financial benefit for the fast

food industry because if there is less water in potatoes, they absorb less oil as they

are French-fried, and can be advertised as lower in fat. The experiment will be an

assay to determine how much starch is present in potato cell extract. The

experiments will be carried out in volumes of no more than 3 ml with 1 gm of

ground potato added to each test. The product of the degradation of the starch will

be maltose, which will be tested for under standard conditions given by Sigma-

Aldrich. You will be in charge of preliminary testing on 1000 recombinant potato

plants that may be used to produce seed for growing commercial crops. It is an

important test and although costs must be kept to a minimum, good science is

what is most important. Either go online to the Sigma-Aldrich website or to their

printed catalogue (available in the lab prep area) and decide between products

A6814 and A6380 -amylase for use in this project. Remember to consider the

experimental application and the cost. Explain your choice.

8. Optional question: Do some research on your own and define Isoelectric Point

(PI). In ion exchange chromatography, how is the pH of the column packing

material you use related to the PI of the protein you are isolating?

lab 4 protein purification by ion exchange student...

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