gelatin zymography for detection of matrixmetalloproteinase-2 and -9 (mmp-2, mmp-9) from myocardiam...

8/11/2019 Gelatin Zymography for Detection of Matrixmetalloproteinase-2 and -9 (MMP-2, MMP-9) From Myocardiam Samples

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Practical course: Basic biochemical methods and

ischemic heart models

Gelatin zymography for detection of matrix-metalloproteinase-2 and -9 (MMP-2, MMP-9) from

myocardiam samples

A practical manual

Krisztina Kupai, PharmD, PhD

2011

Supported by: HURO/0901/069/2.3.1

HU-RO-DOCS


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Table of content

INTRODUCTION OF MATRIX METALLOPROTEINASES (MMPS) .............. 3

INTRODUCTION OF ZYMOGRAPHY ............................................................... 4

SAMPLES FOR ZYMOGRAPHY ................................................................ ....... 6

Isolation procedure of rat heart to detect tissue MMP-2 ............................... 6

FLOW CHART OF GELATIN ZYMOGRAPHY ................................ ................. 7

SAMPLE HOMOGENIZATION ................................... ..................................... ... 7

PROTEIN CONCENTRATION MEASUREMENT FROM SUPERNATANTWITH BCA KIT AND SAMPLE MIXING WITH LOADING BUFFER .............. 8

PREPARATION OF SEPARATING GEL ........................................................ 10

PREPARATION OF STACKING GEL ............................................................. 12

LOADING SAMPLES: ....................................................................................... 12

RUNNING OF GELS ..................................................... .................................... 13

WASHING (renaturation) AND INCUBATION OF THE GELS .................... 14

GEL STAINING ................................ ................................. ................................. 14

REPRESENTATIVE GELATIN ZYMOGRAM GEL ........................................ 16

APPENDIX - PREPARING SOLUTIONS ........................................................ 17

REFERENCES ................................ ................................... ................................ 20


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The full-length MMP-2 can be activated in two ways. Proteolyticactivation of MMP-2 by MT1-MMP/TIMP or by other proteases occursby removal of the autoinhibitory propeptide domain (left arrow)resulting in an active truncated MMP-2. The presence of oxidativestress (ONOO) and cellular glutathione (GSH) causes the S-gluathiolation of the critical cysteine residue in the propeptide domain,disrupting its binding to the catalytic Zn 2+ ion, resulting in an active full-length enzyme.MMP-2 also known as 72kDa collagenase IV or gelatinase A issynthesized as a 631 amino acid proenzyme which is activated bycleavage of the first 80 amino acids.MMP-9 or gelatinase B full length is 92 kDa (proenzyme,) and cleavedactive enzyme is 84 kDa.

ONOO: peroxynitrite; TIMP: tissue inhibitor of metalloproteinase

INTRODUCTION OF ZYMOGRAPHYZymography is known as an electrophoretic technique,

commonly based on sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE), which contains a substratecopolymerized within the polyacrylamide gel matrix (e.g gelatin), forthe detection of an enzymatic activity. Samples are normally prepared

by the standard SDS-PAGE treatment buffer, under non-reducingconditions, i.e. absence of heating and reducing agent [2-mercaptoetanol, dithiothreitol (DTT)]. After the electrophoretic run, theSDS is soaked out from the gel (zymogram) by incubation in a non-buffered Triton X-100 (or similar detergent), followed by incubation inan appropriate activation buffer, for an optimized length of time andtemperature, depending on the type of enzyme being assayed and thetype of substrate being degraded. The zymogram is subsequentlystained, and areas of digestion are distinguished. Though manydifferent types of zymography exist (according to the type of enzyme),not all are possible to mention in this protocol.

For the specific case of proteases (MMP-2, MMP-9) gelatin isone of the most frequently used substrate. In this case, visualization ofthe proteolytic activity appears as clear bands over a deep bluebackground, after Coomassie staining.


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Source: Recent Patents on Biotechnology 2009, 3, 175-184

Schematic overview of some zymographic techniques. (A) 1-Dzymography consists in a SDS-PAGE, with a co-polymerizedsubstrate. After run and enzyme activation, active bandscorresponding to the enzymes are seen as white bands with a bluebackground. ( B). For the 2-D zymogram, the sample must be first

submitted to IEF and then to SDS-PAGE with the co-polymerizedsubstrate. After run and incubation, white spots are evidence ofenzymatic activity. ( C) The spots obtained by 2-DZ, may be furtheranalyzed by MALDI-TOF/MS, in order to identify the enzyme. ( D) In-situ zymography (ISZ) is performed directly on tissue samples,allowing cellular localization of the enzymatic activity. ( E) Real-timezymography (RTZ) allows continuous detection of the enzymaticactivity over time. ( F) Multiple layer substrate zymography (MLSZ)allows the simultaneous detection of different kind of enzymes fromone gel. A gel (1) is run with the sample and then furtherelectrotransferred to several zymograms (2, 3 and 4) containingdifferent substrates. The net result is that with one run, it ispossible to detect different enzymes. ( G) Reverse zymographyconsists in incorporating not only a substrate to the gel, but also theenzyme. Thus, it is possible to detect enzyme inhibitors, visible asdark bands on a light background.


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SAMPLES FOR ZYMOGRAPHYZymography is suitable for analysis of MMPs in complex

biological fluids (serum, synovial fluid) and tissue extracts (heart, liver,

kidney, spleen, etc.).Isolation procedure of rat heart to detect tissue MMP-2

Male Wistar were fed a standard rat chow diet. At age of 8weeks hearts were excised and perfused on a Langendorff perfusionapparatus for 10 min to eliminate the blood from the heart. At the endof 10 min washing period hearts were freeze-clamped and crushed atliquid N 2.

Isolated Langendorff rat heart preparation : As shown infigures, this involves the cannulation of the aorta which is thenattached to a reservoir containing oxygenated perfusion fluid. Thisfluid is then delivered in a retrograde direction down the aorta either ata constant flow rate (delivered by an infusion or roller pump) or aconstant hydrostatic pressure (usually in the range of 60-100mmHg).In both instances, the aortic valves are forced shut and the perfusionfluid is directed into the coronary ostia thereby perfusing the entireventricular mass of the heart, draining into the right atrium via thecoronary sinus.


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FLOW CHART OF GELATIN ZYMOGRAPHY

SAMPLE HOMOGENIZATIONProcedure:

1. Weigh out 30 mg pulverized heart tissue and addhomogenization buffer 1:4 ratio (30 mg tissue+120 lhomogenization buffer)

2. Homogenize the sample 3x10 sec with Pestle homogenizatoron ice

3. Centrifure the samples on 4 C and 5000 g4. Collect the supernatant which contains the MMPs

Pestle homogenizator


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PROTEIN CONCENTRATION MEASUREMENT FROMSUPERNATANT WITH BCA KIT AND SAMPLE MIXING WITH

LOADING BUFFER

I. Measure the protein concentration of supernatant with BCA(bicinchoninic acid ) method

In case of heart samples must be diluted (20x) before proteinmeasurement. Follow the instuction of Pierce BCA Protein Assay Kit(Cat No:23225)

Principle of protein concentration measurement with BCA

II. Calculete the loaded protein amount and mix the the sampleswith loading buffer:

1. We have homogenized a heart sample aftercentrifuge=supernatant and need to load 20 g protein per lane. Sincewe load 20 L per lane, this means that the final protein concentrationof sample needs to be 20 g/20 L.2. In case we want to load a sample only once, it is enough toprepare 1.5 volume of one load (30 L), which means that we shouldadd 15 L non-reducing loading buffer.3. The remaining 15 L should contain 1.520 g=30 g protein.Therefore, volume of the sample will be: V 1=C 1/30, where C 1 is theprotein concentration of your sample.


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4. Then we should add homogenization buffer to dilute thesample. Volume of which will be: V 2=15-V 1.

Taken together:

Loading volume: 20 LLoaded protein: 20 gPrepared volume: 30 L (1.5 load)Loading buffer: 15 LSample: V 1=C 1/30Homogenization buffer: V 2=15-V 1


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PREPARATION OF SEPARATING GELThe final acrylamide concentration in the separating gel (8%) forMMP-2 and MMP-9

STOCK SOLUTIONS VOLUME

30% acrylamide/ 0.8%bisacrylamide

4.0 ml

1.5 MTris HCl, pH 8.8 3.75 ml

ddH 2O 5.75 ml

Gelatin Solution

(20 mg/mL, 1 % w/v SDS)

1.5 ml

10% w/v Ammonium PersulfateSolution (APS)

50 L

TEMED 10 L

MATERIALS: 25 mL glass bakerpipetteselectrophoresis unit (casting mount, glasses,spacers, combs, seals)butanolwipe, blotting paper

PROCEDURE:

1. Prepare gelatin solution (See:Appendix).2. Assemble electrophoresis unit. In order to avoid leakage, ensure

that spacers and glass plates are perfectly aligned.3. Mark desired level of separating gel on unit (use comb).4. Mix 30% acrylamide/0.8% bisacrylamide solution with Tris HCl, pH

8.8, gelatin solution and ddH 2O.5. Add 10% APS solution and TEMED to the mix quickly.


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6. Swirl to mix. Avoid bubbling. USE IMMEDIATELY as polymerizationprocess has begun.

7. Using a pipette, pour a small amount into sandwich plates andwatch for leakage. No leaks? - continue to fill to 1 mm above line.

8. Gently add butanol along top to remove bubbles (avoid shootingthe butanol).

9. Allow gels to polymerize (approximately 20 minutes at 25 C). Usethis time to prepare stacking gel (without adding TEMED and 10%

APS) TIP: leave pipette in the left over separating gel, when it ispolymerized you will be able to lift it with the pipette.

10.A layer of H 2O on top of the gel will be visible when polymerizationis complete. Drain this layer from the unit with a small stripe ofblotting paper.

11.Prepare the stacking gel

Combs and spacers for gel

Preparation of separating gel(Source:http://www.soonersci.com/catalog/page21.html)


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PREPARATION OF STACKING GELMATERIALS: 25 mL glass baker

Pipettes

STOCK SOLUTIONS VOLUME

30% acrylamide/ 0.8%bisacrylamide

0.65 ml

0.5 M Tris HCl pH 6.8 1.25 ml

ddH 2O 3.05

10% SDS 50 L

10% w/v AmmoniumPersulfate Solution (APS)

25 L

TEMED 8 L

PROCEDURE:

1. Mix 30% acrylamide/0.8% bisacrylamide solution with Tris HCl, pH6.8 and ddH 2O.

2. Add 10% SDS, 10% APS and TEMED.3. Swirl to mix. Avoid bubbling. USE IMMEDIATELY as polymerization

process has begun.4. Place comb in units and then use pipettes to pour stacking gel.5. Place stacking gel solution on ice and continue to top up the gel

until polymerization is complete.

LOADING SAMPLES:MATERIALS: 1000 mL running buffer

20 L pipette


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PROCEDURE:

1. Prepare and cool down running buffer.2. Map out gel lanes for sample loading.3. When gel is polymerized, remove combs by pulling straight up.4. Remove gel plates and snap onto electrode assembly.5. Fill up the lower and the upper buffer container with running buffer.6. Load samples.7. Load protien molecular weight and purified MMP-2 as an internal

standard

RUNNING OF GELS

1. Connect electrodes properly2. Set voltage at 90 V.3. For 8% gel run for 0.5 hour after dye front disappears. (The exact

duration of electrophoresis will vary from person to person.)4. Use this time to prepare Triton X-100 solution and incubation

buffer.

Electrophoretic tank&steps electrophoresis(Source:http://www.topac.com/vertical_casting.html)

1. With plates in position place central running module in thecasting base.2. Turn both cams to pull module down onto silicone sealing units.3. Pour gel between plates.4. Place appropriate comb between plates and allow gels to set.5. After carefully removing combs release module from castingbase.6 Place the module in the outer buffer tank and fill the two bufferchambers.


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7. Load sample and replace lid.8. Unit is now ready for electrophoresis.

Sample separation on SDS-gelatin gel after electrophoresis

WASHING (renaturation) AND INCUBATION OF THE GELSMATERIALS: 1000 mL 2.5% v/v Triton X-100 aqueous

solution1000 mL incubation buffer1000 mL dd H 2Owater bath (set at 37 oC)

PROCEDURE:

1. Set incubator at 37 oC.2. Disassemble gel apparatus.3. Cut the bottom left corner of gel #1, and both the top and bottom

left corners for gel #2. (Ensure that the gel is oriented correctly sothat you dont accidentally cut the right side corner!)

4. Wash gels for 40 min in 300-500 mL 2.5% Triton X-100 solution atroom temperature.

5. Place gels in incubation buffer (500 mL per gel).

6. Incubate gels for 20 h at 37oC.

GEL STAINING

MATERIALS: 0.05% Coomassie Brilliant Blue G-250 solutiondestaining solution


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PROCEDURE:

1. Even before staining, gelatinolytic activity should be visible (holdthe gel up against a dark background to visualize).

2. Put gels in Coomassie Brilliant Blue solution. Place on shaker for 1- 2 hours.

3. Put gels in destaining solution. Place on shaker for 2 hours4. Scan the scan5. Gelatinolytic activities should be detected as transparent bands

against the blue background of Coomassie Brilliant Blue stainedgelatin.

Pour the in Coomassie Brilliant Blue to gels

Rocking the freezer box on the tilt table will

ensure that the gel gets evenly stained.

Destaining of the gel will remove any

Coomassie blue dye that is not bound toprotein.


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REPRESENTATIVE GELATIN ZYMOGRAM GEL

Zymogram gel at the end of procedure: gelatinolytic activities shouldbe detected as transparent bands against the blue background ofCoomassie Brilliant Blue stained gelatin

MMPs are initially secreted as an inactive proenzyme orzymogen, which supposely have no gelatinolytic activity. So how thezymography works to show the pro-MMP2, pro-MMP9 binds, since theproenzymes cannot digest the gelatin?

The enzymes are separated in denaturing conditions (SDS),refolded in Triton (to remove the SDS), then incubated. The zymogensforms are activated by this process of denaturation and renaturation,and so may be visualized in the zymogram.In this way, they migrate according to the molecular weight (pro-formmigrate less than the active form) and both are seen at the gel.This is the main advantage of zymography, you can visualize bothforms.


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APPENDIX - PREPARING SOLUTIONS

I.Separating Gel Solution 1.5 M Tris HCl, pH 8.8

BIO-RAD 161-0798

II,Stacking Gel Solution 0.5 M Tris HCl/SDS, pH 6.8

BIO-RAD 161-0799

III.30% Acrylamide / 0.8% Bisacrylamide

**NOTE: Acrylamide monomer should be weighed out in fume hoodas it is neurotoxic.

BIO-RAD 161-0156

IV.10% (w/v) Ammonium Persulfate Solution

MATERIALS: 100 mg ammonium persulfate (Sigma A-6761)

1 mL ddH 2O

1.Dissolve 100 mg of APS in 1 mL ddH 2O.

NOTE - should be prepared freshly

V.Gelatin Solution

MATERIALS: 100 mg gelatin (type A, from porcine skin, SIGMAcat # G-8150)4.5 mL ddH 2O0.5 mL 10 % w/v SDS aqueous solution


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glass beaker + stir bar

1. Add gelatin to the H 2O.2. Gently heat solution until gelatin dissolves (beaker will be warm to

touch).3. Add 10% w/v SDS aqueous solution to reach final desired volume.

VI. _Running Buffer

(25mM Tris, 192 mM glycine, 0.1% SDS, pH=8.3)

BIO-RAD 161-0732 or Pierce 28378

Or prepare it:

MATERIALS: 2000 mL graduated cylinder

28.83 g glycine

6.0 g Tris base

2.0 g SDS

2000 mL ddH 2O

1. Dissolve Tris base and glycine in 1000 mL of ddH 2O.2. Bring solution to 1950 mL with ddH 2O.3. Add SDS.4. Bring solution to 2000 mL total volume with ddH 2O.

VII.Non reducing Loading (Sample) Buffer

Bio-Rad 161-0764

VIII.Triton X-100 solution (2.5% v/v)

MATERIALS: 1000 mL glass beaker + stir bar


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25 mL Triton X-100

1000 mL ddH 2O

1. Add 25 mL of Triton-X solution SLOWLY to 900 mL of ddH 2O whilecontinuosly mixing. (NOTE: Triton X-100 is a viscous fluid pourinto solution slowly )

2. Bring total volume up to 1000 mL with ddH 2O.

IX.Incubation Buffer (50 mM Tris HCl, 0.15 M NaCl, 10 mM CaCl 2)

MATERIALS: 8.766 g NaCl

1.47g CaCl 2*2H2O

6.057 g Tris base

0.5 g NaN 3 (weigh in fumehood toxic )

1000 mL ddH 2O

1. Add NaCl, CaCl 2, Tris base, and 0.5 g NaN 3 to 1000 mL of ddH 2O.2. Adjust to pH 7.8-8.0 with cc. HCl.

X.Coomassie Brilliant Blue (0.05%)

MATERIALS: 500 mL glass beaker

250 mg Coomassie Brilliant Blue G-250(Sigma B-1131)

125 mL methanol

50 mL glacial acetic acid

325 mL ddH 2O


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XI.Destaining Solution

MATERIALS: 1000 mL glass beaker

40 mL methanol

80 mL acetic acid

880 mL ddH 2O

XII.Homogenization Buffer

MATERIALS: 500 ml ddH 2O

0.335 g (50 mM) Tris base (Merck 648310-500GM)

1 ml (0.5%) Triton

1. In 500 ml beaker dissolve compounds in 500 ml dd H 2O.2. Adjust to pH 7.4 with 1 M HCl.3. Aliquot into 15 ml Falcon tubes.

REFERENCES

1. Kupai K.; J Pharmacol Toxicol Methods. 2010 Mar-

Apr;61(2):205-9; Matrix metalloproteinase activity assays:Importance of zymography.

2. Laemmeli,E.K.; Nature. 227:680-685, 1970; Cleavage ofstructural proteins during the assembly of the head ofbacteriophage T4.


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3. Jeff Wilkesman, Liliana Kurz, Recent Patents on Biotechnology2009, 3, 175-184; Protease Analysis by Zymography: A Reviewon Techniques and Patents

4. AK Chow, J Cena, R Schulz, British Journal of Pharmacology(2007) 117; Acute actions and novel targets of matrixmetalloproteinases in the heart and vasculature

gelatin zymography for detection of matrixmetalloproteinase-2 and -9 (mmp-2, mmp-9) from myocardiam...

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