1. Sodium dodecyl sulfate- Polyacrylamide gel electrophoresis (SDS-PAGE) Irene Goh Rosarine Metusela

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

3. 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

4. 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

5. 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.

6. 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)

7. 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

8. 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

9. 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

10. 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

11. 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.

12. Results and discussion  The results discussed here is, the sample results which was provided by the supervisor

13. Results and discussion Protein Standard Theoretical MW log10 MW Distance migrate d (cm) Relative distance Aprotinin, bovine lung 6,5003.8129133571.650.113793103 a-lactalbumin, bovine milk 14,2004.1522883443.550.244827586 Trypsin inhibitor 20,1004.3031960574.050.279310345 Tyrpsinogen, bovine pancrease 24,0004.3802112424.550.313793103 Carbonic anhydrase 29,0004.4623979984.900.337931034 Glyceraldehyde-3- phosphatedehydrogenase 36,0004.5563025015.850.403448276

14. Results and discussion Protein Standard Theoreti cal MW log10 MW Distance migrated (cm) Relative distance Glutamic dehydrogenase, bovine liver 55,000 4.740362 689 6.600.455172414 Albumin, bovine serum 66,000 4.819543 936 7.650.527586207 Fructose-6- phosphate kinase 84,000 4.924279 286 8.350.575862069 Phosphorylase b, rabbit muscle 97,000 4.986771 734 8.750.603448276 B-galactosidase, E.coli 116,000 5.064457 989 9.750.672413793 Myosin, rabbit muscle 205,000 5.3117538 61 12.400.855172414 Glutamic dehydrogenase, bovine liver 55,0004.7403626896.600.455172414 Albumin, bovine serum 66,0004.8195439367.650.527586207 Fructose-6- phosphate kinase 84,0004.9242792868.350.575862069 Phosphorylase b, rabbit muscle 97,0004.9867717348.750.603448276 B-galactosidase, E.coli 116,0005.0644579899.750.672413793 Myosin, rabbit muscle 205,0005.31175386112.400.855172414

15. Results and discussion

16.  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

17. Results and discussion Sample lanedistance(cm) relative distancelog10 MrMr (Da) b (i)2.50.1724137934.05499225311349.9057 (ii)5.050.3482758624.42252008826455.75061 (iii)7.90.5448275864.83328649268121.85908 c3.10.2137931034.14146939113850.62563 d9.150.6310344835.013447195103144.766 e5.650.3896551724.50899722632284.73497 f4.050.2793103454.27839152518984.16611 g8.950.6172413794.98462148296520.92657 h11.40.7862068975.337736461217638.8693 I4.250.2931034484.30721723820286.97237 j3.70.2551724144.22794652816902.32812 k7.650.5275862074.79725435162698.09577 l4.750.3275862074.37928151923948.67659 Mr => Relative molecular weight of the unknown samples.

18. 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.

19. 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