Sodium dodecyl sulfate-Sodium dodecyl sulfate-Polyacrylamide gel Polyacrylamide gel

electrophoresis (SDS-PAGE)electrophoresis (SDS-PAGE)

Irene Goh Irene Goh

Rosarine MetuselaRosarine Metusela

ObjectivesObjectives

To use the SDS PAGE analytical procedure to To use the SDS PAGE analytical procedure to identify and/or isolate the following proteins: identify and/or isolate the following proteins:

••Ovalbumin Ovalbumin

••Casein Casein

••Gluten Gluten To be able to understand the principles of gel To be able to understand the principles of gel

electrophoresis electrophoresis To apply and follow safety procedures while To apply and follow safety procedures while

carrying out the experiment carrying out the experiment

What is SDS-PAGE?What is SDS-PAGE?

Based on the migration of charged Based on the migration of charged molecules in an electric fieldmolecules in an electric field

Separation techniqueSeparation technique Uses the Polyacrylamide gel as a “support Uses the Polyacrylamide gel as a “support

matrix”. The matrix inhibits convective matrix”. The matrix inhibits convective mixing caused by heating and provides a mixing caused by heating and provides a record of the electrophoretic run.record of the electrophoretic run.

Polyacrylamide is a porous gel which acts Polyacrylamide is a porous gel which acts as a sieve and separates the molecules as a sieve and separates the molecules

Role of SDSRole of SDS

Denatures proteins by wrapping around the Denatures proteins by wrapping around the polypeptide backbone. polypeptide backbone.

SDS binds to most proteins in amount roughly SDS binds to most proteins in amount roughly proportional to molecular weight of the protein-proportional to molecular weight of the protein-about one molecule of SDS for every two amino about one molecule of SDS for every two amino acids (1.4 g SDS per gram of protein) (Lehninger acids (1.4 g SDS per gram of protein) (Lehninger Principles of Biochemistry). Principles of Biochemistry).

In doing so, SDS creates a large negative charge In doing so, SDS creates a large negative charge to the polypeptide in proportion to its length to the polypeptide in proportion to its length

Role of SDS (cont…)Role of SDS (cont…)

SDS also disrupts any hydrogen bonds, blocks many SDS also disrupts any hydrogen bonds, blocks many hydrophobic interactions and partially unfolds the protein hydrophobic interactions and partially unfolds the protein molecules minimizing differences based on the secondary molecules minimizing differences based on the secondary or tertiary structure or tertiary structure

Therefore, migration is determined Therefore, migration is determined notnot by the electrical by the electrical charge of the polypeptide, but by charge of the polypeptide, but by molecular weightmolecular weight. .

The rate at which they move is inversely proportional to the The rate at which they move is inversely proportional to the molecular massmolecular mass

This movement is then used to determined the molecular This movement is then used to determined the molecular weight of the protein present in the sample. weight of the protein present in the sample.

Procedure: materialsProcedure: materials

1.A Mighty Small II, SE 260 Mini-Vertical Gel 1.A Mighty Small II, SE 260 Mini-Vertical Gel Electrophoresis Unit Electrophoresis Unit

2.0.5 TrisCl, pH 6.8 solution 2.0.5 TrisCl, pH 6.8 solution 3.10% SDS solution 3.10% SDS solution 4.Sample treatment buffer 4.Sample treatment buffer 5.SDS glycine running buffer 5.SDS glycine running buffer 6.β-Mercaptoethanol solution 6.β-Mercaptoethanol solution 7.Brilliant Blue R concentrate 7.Brilliant Blue R concentrate 8.Destaining solution 8.Destaining solution 9.Precast polyacrylamide separating gel 9.Precast polyacrylamide separating gel 10.Fine tipped microsyringe 10.Fine tipped microsyringe 11.Protein samples (ovalbumin, casein, and gluten) 11.Protein samples (ovalbumin, casein, and gluten)

Procedure: solutionsProcedure: solutions

0.5M TrisCl, pH 6.8 (4X Resolving gel 0.5M TrisCl, pH 6.8 (4X Resolving gel buffer) buffer)

10% SDS solution 10% SDS solution 2X Sample treatment buffer2X Sample treatment buffer SDS glycine running buffer SDS glycine running buffer Destaining solution Destaining solution

Procedure: electrophoresis unitProcedure: electrophoresis unit

Initial preparation-wash the unitInitial preparation-wash the unit Preparing the gel sandwich(es): Preparing the gel sandwich(es):

– ensure that the plates are completely polymerized ensure that the plates are completely polymerized before loadingbefore loading

– Install the gel sandwhich(es) into the unit before loading Install the gel sandwhich(es) into the unit before loading any of the protein samples.any of the protein samples.

Loading the protein samples:Loading the protein samples:– Dry sample: add equal volumes of treatment buffer Dry sample: add equal volumes of treatment buffer

solution, and deionised water to achieve the required solution, and deionised water to achieve the required concentration. Heat in a tube, in boiling water for 90 concentration. Heat in a tube, in boiling water for 90 seconds seconds

Procedure: electrophoresis unitProcedure: electrophoresis unit

Fill upper buffer chamber with running bufferFill upper buffer chamber with running buffer Using a fine-tipped microsyringe, load the Using a fine-tipped microsyringe, load the

treated protein samples into the wells so treated protein samples into the wells so that the volume in each well is raised by that the volume in each well is raised by 1mm 1mm

Fill the lower buffer chamberFill the lower buffer chamber

Procedure: running the gelProcedure: running the gel

Place the safety lid on Place the safety lid on beforebefore plugging in plugging in the leads of the unit to the power supply.the leads of the unit to the power supply.

Run the gel at 20mA per gel, using a Run the gel at 20mA per gel, using a constant current constant current

When it reaches the bottom of the gel, the When it reaches the bottom of the gel, the run is completerun is complete

Turn off the power supply, and disconnect Turn off the power supply, and disconnect the leads, before removing the safety lid the leads, before removing the safety lid

Procedure: running the gelProcedure: running the gel

Carefully remove the gel(s) from the platesCarefully remove the gel(s) from the plates Lay it into a tray of staining solution for Lay it into a tray of staining solution for

about 10 minutes. about 10 minutes. Remove the gel carefully and place it in Remove the gel carefully and place it in

between two layers of transparencies, cut between two layers of transparencies, cut along the edges of the gel and analyse the along the edges of the gel and analyse the results. results.

Results and discussionResults and discussion

The results discussed here is, the sample The results discussed here is, the sample results which was provided by the results which was provided by the supervisor supervisor

Results and discussionResults and discussion

Protein Protein StandardStandard

Theoretical Theoretical MWMW log10 MWlog10 MW

Distance Distance migratemigrated (cm)d (cm)

Relative Relative distancedistance

Aprotinin, bovine lungAprotinin, bovine lung 6,5006,500 3.8129133573.812913357 1.651.65 0.1137931030.113793103

a-lactalbumin, bovine milka-lactalbumin, bovine milk 14,20014,200 4.1522883444.152288344 3.553.55 0.2448275860.244827586

Trypsin Trypsin inhibitorinhibitor 20,10020,100 4.3031960574.303196057 4.054.05 0.2793103450.279310345

Tyrpsinogen, bovine Tyrpsinogen, bovine pancreasepancrease 24,00024,000 4.3802112424.380211242 4.554.55 0.3137931030.313793103

Carbonic Carbonic anhydraseanhydrase 29,00029,000 4.4623979984.462397998 4.904.90 0.3379310340.337931034

Glyceraldehyde-3-Glyceraldehyde-3-phosphatedehydrogenasephosphatedehydrogenase 36,00036,000 4.5563025014.556302501 5.855.85 0.4034482760.403448276

Results and discussionResults and discussion

Protein Protein StandardStandard

TheoretiTheoretical cal MWMW

log10 log10 MWMW

Distance Distance migratedmigrated

(cm)(cm)Relative Relative

distancedistance

Glutamic dehydrogenase, Glutamic dehydrogenase, bovine liverbovine liver 55,00055,000

4.7403624.740362689689 6.606.60 0.4551724140.455172414

Albumin, bovine serumAlbumin, bovine serum 66,00066,0004.8195434.819543

936936 7.657.65 0.5275862070.527586207

Fructose-6- phosphate kinaseFructose-6- phosphate kinase 84,00084,0004.9242794.924279

286286 8.358.35 0.5758620690.575862069

Phosphorylase b, rabbit Phosphorylase b, rabbit musclemuscle 97,00097,000

4.9867714.986771734734 8.758.75 0.6034482760.603448276

B-galactosidase, B-galactosidase, E.coliE.coli 116,000116,0005.0644575.064457

989989 9.759.75 0.6724137930.672413793

Myosin, rabbit muscleMyosin, rabbit muscle 205,000205,0005.3117535.311753

861861 12.4012.40 0.8551724140.855172414

Glutamic dehydrogenase, bovine liverGlutamic dehydrogenase, bovine liver 55,00055,000 4.7403626894.740362689 6.606.60 0.4551724140.455172414

Albumin, bovine serumAlbumin, bovine serum 66,00066,000 4.8195439364.819543936 7.657.65 0.5275862070.527586207

Fructose-6- phosphate kinaseFructose-6- phosphate kinase 84,00084,000 4.9242792864.924279286 8.358.35 0.5758620690.575862069

Phosphorylase b, rabbit musclePhosphorylase b, rabbit muscle 97,00097,000 4.9867717344.986771734 8.758.75 0.6034482760.603448276

B-galactosidase, B-galactosidase, E.coliE.coli 116,000116,000 5.0644579895.064457989 9.759.75 0.6724137930.672413793

Myosin, rabbit muscleMyosin, rabbit muscle 205,000205,000 5.3117538615.311753861 12.4012.40 0.8551724140.855172414

Results and discussionResults and discussion

Standard curves for proteins with known molecular weights

y = 0.4785x - 1.7679

R20.9672 =

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 1 2 3 4 5 6

log10 MW

Rela

tive M

igra

tio

n (

cm

)

Results and discussionResults and discussion

the relationship between the logarithm of the the relationship between the logarithm of the standards and the relative distance travelled by standards and the relative distance travelled by each protein through the gel is linear each protein through the gel is linear

The equation of the line was obtained and used to The equation of the line was obtained and used to calculate the relative molecular weights (Mr) of calculate the relative molecular weights (Mr) of the samples in lanes b-l of the gelthe samples in lanes b-l of the gel

x = (y + 1.7679)/0.4785x = (y + 1.7679)/0.4785x – Mrx – Mry – Relative distance travelled by the sample in y – Relative distance travelled by the sample in

centimetrescentimetres

Results and discussionResults and discussionSample lane distance(cm)

relative distance log10 Mr Mr (Da)

b (i) 2.5 0.172413793 4.054992253 11349.9057

(ii) 5.05 0.348275862 4.422520088 26455.75061

(iii) 7.9 0.544827586 4.833286492 68121.85908

c 3.1 0.213793103 4.141469391 13850.62563

d 9.15 0.631034483 5.013447195 103144.766

e 5.65 0.389655172 4.508997226 32284.73497

f 4.05 0.279310345 4.278391525 18984.16611

g 8.95 0.617241379 4.984621482 96520.92657

h 11.4 0.786206897 5.337736461 217638.8693

I 4.25 0.293103448 4.307217238 20286.97237

j 3.7 0.255172414 4.227946528 16902.32812

k 7.65 0.527586207 4.797254351 62698.09577

l 4.75 0.327586207 4.379281519 23948.67659

Mr => Relative molecular weight of the unknown samples.

Results and discussionResults and discussion

From the molecular weights obtained for the From the molecular weights obtained for the proteins to be analysed in the experiment:proteins to be analysed in the experiment:– Cassein = 24,000 DaCassein = 24,000 Da– Ovalbumin = 46,000 DaOvalbumin = 46,000 Da– Gluten = 20,000 – 11,000,000 DaGluten = 20,000 – 11,000,000 Da

It would be expected that the relative It would be expected that the relative molecular weights of these proteins, would molecular weights of these proteins, would be close their respective theoretical values be close their respective theoretical values shown above.shown above.

ConclusionConclusion

SDS PAGE is a useful method for SDS PAGE is a useful method for separating and characterising proteins, separating and characterising proteins, where a researcher can quickly check the where a researcher can quickly check the purity of a particular protein or work out the purity of a particular protein or work out the different number of proteins in a mixture.different number of proteins in a mixture.

Since we did not obtain results for the Since we did not obtain results for the experiment, experiment, – we have to rely on sample results we have to rely on sample results – Cannot validate the experimental technique Cannot validate the experimental technique