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: OvalbuminCaseinGluten  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  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

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

Results and discussion Protein Standard Theoreti cal MW log10 MW Distance migrated (cm) Relative distance Glutamic dehydrogenase, bovine liver 55, Albumin, bovine serum 66, Fructose-6- phosphate kinase 84, Phosphorylase b, rabbit muscle 97, B-galactosidase, E.coli 116, Myosin, rabbit muscle 205, Glutamic dehydrogenase, bovine liver 55, Albumin, bovine serum 66, Fructose-6- phosphate kinase 84, Phosphorylase b, rabbit muscle 97, B-galactosidase, E.coli 116, Myosin, rabbit muscle 205,

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

Results and discussion Sample lanedistance(cm) relative distancelog10 MrMr (Da) b (i) (ii) (iii) c d e f g h I j k l 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