A report on an experiment I did of doing electrophoresis with proteins

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2 A. A note on the chemical solutions used Some of the standard acronyms for chemicals that are used are, • “Acrylamide Mix” is a 1 : 29 mixture of N, Nmethylene bis-acrylamide and Acrylamide • “SDS” is Sodium Dodecyl Sulphate • “APS” is Ammonium per-Sulphate (always stored at −20 ◦ C) • “TEMED” is N,N,N’,N’-Tetramethylethyl-enediamine (always stored at 4 ◦ C) All the autoclaving in this experiment are done at 121 ◦ C for 15minutes at 103kP a of pressure in an autoclaving apparatus made by Hospharma. The mixture of chemicals required to make 15ml of Separating Gel are, • H 2 O −→ 6.9ml • 30%Acrylamide Mix −→ 4.0ml • 1.5M T ris − Hcl(pH 8.8) −→ 3.8ml • 10% SDS −→ 0.15ml • 10% AP S −→ 0.15ml • T EMED −→ 0.009ml The mixture of chemicals required to make 5ml of Stacking Gel are, • H 2 O −→ 3.4ml • 30%Acrylamide Mix −→ 0.83ml • 1M T ris − Hcl(pH 8.8) −→ 0.63ml • 10% SDS −→ 0.05ml • 10% AP S −→ 0.05ml • T EMED −→ 0.005ml To make 2l of the Electrophoresis Buffer that will act as the conductor for the current during electrophoresis one uses the following mixture, • Trizma Base (a.k.a Tris), which is mainly C 4 H 11 NO 3 → 12g • Glycine → 57.6g • SDS → 2g • Autoclaved water is added to make up the volume to 2l and pH adjustment is not done. To make 50ml of the Sample Buffer (DTT dye) one has to mix the following, • 0.6M solution of Tris in HCl at pH 6.8 → 5ml • SDS → 0.5g • Sucrose → 5g • β − Mercaptoethanol → 0.25ml • 0.5% Bromophenol Blue → 5ml • Autoclaved water is added to make up the solution to 50ml
3 To make Staining Solution one mixes, • 0.1%(v/v) Coomassie brilliant blue R250 • 50%(v/v) Methanol • 10%(v/v) Glacial Acetic acid. To make the Destaining Solution one mixes, • 10% Methanol • 7% Glacial Acetic acid To make many of these solutions the masses of the chemicals needed to be measured and they were done on these two machines manufactured by Sartorius, • One was the model CP 1245 and it had a maximum load capacity of 120g with a least count of 10 −4 g • The other was of model BL1500S and it had a maximum load capacity of 1500g with a least count of 10 −2 g The following are the steps in setting the gels, B. Setting the gels • Before one starts making the solutions one sets up the two pairs of glass plates on the rack in a particular orientation as marked on the plates. Each of these glass plates is about 1mm think and specially made to be as smooth as possible and are very fragile. The larger plat is of dimensions about 10cm × 8.2cm and the smaller one is of size about 10cm × 7.3cm. There are two about 1mm thick strips of opaque glass of sizes almost 8.2cm × 9mm stuck on the two sides of the larger glass. This ensures a gap of 1mm between the two glass plates in which the gel will be polymerized. • One makes the solutions of the separating and the stacking gel in separate containers. I made them in discarded 50ml aliquots manufactured by Falcon. • Except APS and TEMED one mixes in all the chemicals in the two compositions given above. • APS needs to be freshly prepared or one that has been stored at −20 ◦ C and just taken out. • Since the separating gel will be made first hence for now one puts the APS in the separating solution only and mixes the TEMED also in only that one. • As soon as the APS and TEMED are poured into the separating solution one immediately transfers them between one of the pairs of glass-plates through a pipette tip and the tip should be moved all along the edge through out to ensure that the fluid flows in uniformly between the glass plates. Any non-uniformity will result in nucleation centers for the polymerization process and make the gel non-homogeneous. • One stops pouring the solution when it reaches an height of about 1cm below the edge of the plates (such that the gap is larger than the depth of the comb so that the protein solutions have to pass through some stacking gel before entering the separating gel andin the process they undergo isoelectric focussing through the process of isotacphoresis.) One pours butanol on the top of it through another pipette. This is immiscible with the rest of the solution and floats on the top helping to smoothen the gel surface and also acts as a protecting layer from the oxidation effects of the atmosphere. • Now one waits for about 30mins for the gel to form. It can sometimes take an hour or a little more depending on humidity and temperature conditions. After about 30mins I keep checking for the formation of the gel by tilting the whole set up at times to see if the butanol layer is hitting a hard boundary on its lower surface. • Once the separating gel is formed one washes out the butanol layer by tilting the container and gently flowing in autoclaved water through the gap and absorbing any further remnant butanol by gently inserting a tissue paper in the gap.
Page 1: SDS-PAGE Electrophoresis Anirbit De
Page 5 and 6: 5 D. Making BSA dilution solution T
Page 7 and 8: • The voltage is maintained till
Page 9 and 10: 9 FIG. 7: The basic idea is of two
Page 11 and 12: 11 H. Image analysis on the gel Aft
Page 13 and 14: 13 FIG. 14: A pot of the points (1
Page 15 and 16: 15 FIG. 18: The above graph shows t
Page 17 and 18: But for a fixed protein if m 0 and
Page 19 and 20: 19 E l ∑i [u i]µ iu z iu = E u

A report on an experiment I did of doing electrophoresis with proteins

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