Sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE)

Irene Goh Rosarine Metusela

Objectives

 To use the SDS PAGE analytical procedure to identify and/or isolate the following proteins: •Ovalbumin •Casein •Gluten  To be able to understand the principles of gel electrophoresis  To apply and follow safety procedures while carrying out the experiment

What is SDS-PAGE?

 Based on the migration of charged molecules in an electric field   Separation technique Uses the Polyacrylamide gel as a “support matrix”. The matrix inhibits convective mixing caused by heating and provides a record of the electrophoretic run.

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

Role of SDS

   Denatures proteins by wrapping around the polypeptide backbone. SDS binds to most proteins in amount roughly proportional to molecular weight of the protein about one molecule of SDS for every two amino acids (1.4 g SDS per gram of protein) (Lehninger Principles of Biochemistry). In doing so, SDS creates a large negative charge to the polypeptide in proportion to its length

Role of SDS (cont…)

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

not

by the electrical charge of the polypeptide, but by

molecular weight

. The rate at which they move is inversely proportional to the molecular mass This movement is then used to determined the molecular weight of the protein present in the sample.

Procedure: materials

           1.A Mighty Small II, SE 260 Mini-Vertical Gel Electrophoresis Unit 2.0.5 TrisCl, pH 6.8 solution 3.10% SDS solution 4.Sample treatment buffer 5.SDS glycine running buffer 6.β-Mercaptoethanol solution 7.Brilliant Blue R concentrate 8.Destaining solution 9.Precast polyacrylamide separating gel 10.Fine tipped microsyringe 11.Protein samples (ovalbumin, casein, and gluten)

Procedure: solutions

     0.5M TrisCl, pH 6.8 (4X Resolving gel buffer) 10% SDS solution 2X Sample treatment buffer SDS glycine running buffer Destaining solution

Procedure: electrophoresis unit

   Initial preparation-wash the unit Preparing the gel sandwich(es): – ensure that the plates are completely polymerized before loading – Install the gel sandwhich(es) into the unit before loading any of the protein samples.

Loading the protein samples: – Dry sample: add equal volumes of treatment buffer solution, and deionised water to achieve the required concentration. Heat in a tube, in boiling water for 90 seconds

Procedure: electrophoresis unit

  Fill upper buffer chamber with running buffer Using a fine-tipped microsyringe, load the treated protein samples into the wells so that the volume in each well is raised by 1mm  Fill the lower buffer chamber

Procedure: running the gel

 Place the safety lid on

before

plugging in the leads of the unit to the power supply.

 Run the gel at 20mA per gel, using a constant current  When it reaches the bottom of the gel, the run is complete  Turn off the power supply, and disconnect the leads, before removing the safety lid

Procedure: running the gel

  Carefully remove the gel(s) from the plates Lay it into a tray of staining solution for about 10 minutes.  Remove the gel carefully and place it in between two layers of transparencies, cut along the edges of the gel and analyse the results.

Results and discussion

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

Protein Standard

Results and discussion

Theoretical MW log10 MW Distance migrate d (cm) Relative distance

Aprotinin, bovine lung 6,500 3.812913357

1.65

0.113793103

a-lactalbumin, bovine milk Trypsin inhibitor Tyrpsinogen, bovine pancrease Carbonic anhydrase Glyceraldehyde-3 phosphatedehydrogenase 14,200 4.152288344

20,100 24,000 4.303196057

4.380211242

29,000 36,000 4.462397998

4.556302501

3.55

4.05

4.55

4.90

5.85

0.244827586

0.279310345

0.313793103

0.337931034

0.403448276

Results and discussion

Protein Standard

Glutamic dehydrogenase, bovine liver Albumin, bovine serum Fructose-6- phosphate kinase Phosphorylase b, rabbit muscle B-galactosidase,

E.coli

Myosin, rabbit muscle

Theoreti cal MW

55,000 66,000 84,000 97,000 116,000 205,000

log10 MW

4.740362

689 4.819543

936 4.924279

286 4.986771

734 5.064457

989 5.3117538

61

Distance migrated (cm)

6.60

7.65

8.35

8.75

9.75

12.40

Relative distance

0.455172414

0.527586207

0.575862069

0.603448276

0.672413793

0.855172414

Results and discussion

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0 0

Standard curves for proteins with known molecular weights

1 y = 0.4785x - 1.7679

R 2 0.9672

= 2 3

log10 MW

4 5 6

Results and discussion

   the relationship between the logarithm of the standards and the relative distance travelled by each protein through the gel is linear The equation of the line was obtained and used to calculate the relative molecular weights (Mr) of the samples in lanes b-l of the gel x = (y + 1.7679)/0.4785

x – Mr y – Relative distance travelled by the sample in centimetres

f e g h I j l k

Sample lane

b (i) (ii) (iii) c d

Results and discussion

distance(cm)

2.5

5.05

7.9

3.1

9.15

5.65

4.05

8.95

11.4

4.25

3.7

7.65

4.75

relative distance

0.172413793

0.348275862

0.544827586

0.213793103

0.631034483

0.389655172

0.279310345

0.617241379

0.786206897

0.293103448

0.255172414

0.527586207

0.327586207

log10 Mr

4.054992253

4.422520088

4.833286492

4.141469391

5.013447195

4.508997226

4.278391525

4.984621482

5.337736461

4.307217238

4.227946528

4.797254351

4.379281519

Mr (Da)

11349.9057

26455.75061

68121.85908

13850.62563

103144.766

32284.73497

18984.16611

96520.92657

217638.8693

20286.97237

16902.32812

62698.09577

23948.67659

Mr => Relative molecular weight of the unknown samples.

Results and discussion

  From the molecular weights obtained for the proteins to be analysed in the experiment: – Cassein = 24,000 Da – Ovalbumin = 46,000 Da – Gluten = 20,000 – 11,000,000 Da It would be expected that the relative molecular weights of these proteins, would be close their respective theoretical values shown above.

Conclusion

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

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