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

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8 Some general dimensions of the gel are, F. Some measurements of the gel • The separating gel is generally about 5.5cm × 8.2cm in size and the stacking gel is 1.5cm × 8.2cm with wells along one edge. • The wells in the stacking gel are about 1cm deep and 0.5cm in width, so the proteins pass through about 0.6cm of stacking gel before hitting the separating gel. • The gel is generally 0.1cm in thickness. • The separating gel is about 4.26g in mass and is typically at a density of 0.91g/cc. • The stacking gel is about 0.83g in mass and is typically at a density of 1.03g/cc G. A short note about the chemicals The precise utility of each of the chemicals in the experiment is a extensive question in chemistry and the rational behind the quatities of each to be used is hard to theoretically justiy. Some of the essential physical ideas that go behind it shall be explained at the end in the section on background theory. A basic list of use of the important chemicals are, • Sodium Dodecyle Sulphate is basically a soap which when heated with the protein binds to the amino acids in the protein and hence destabilizes the hydrogen-bonding which renders the protein its structure. This “denatures” the protein by making all of them more or less cylindrical in shape. This ensures that the proteins’s mobility which determines their stacking sequence depends less on the structure and more on the molecular mass. Also, anions of SDS bind to the main peptide chain at a ratio of one SDS anion for every two amino acid residues. This effectively imparts a negative charge on the protein that is proportional to the mass of that protein (about 1.4gSDS/gprotein). This negative charging is essential for the migration of the proteins (precisely the protein- SDS complex) towards the anode during electrophoresis. FIG. 6: SDS • β−Mercaptoethanol, HO −(CH 2 ) 2 −SH is mixed in the protein solution mainly to break the di-sulphide bonds in the protein which also help keep the protein conformation.
9 FIG. 7: The basic idea is of two amino acids bonding to form a peptide. This will exist mostly as a zwitterion in solution and SDS when heated at 100 ◦ C for 5 minutes with proteins will bind at the rate of one SDS anion for every two amino acid residues or approximately as 1.4gSDS/g protein. This not only breaks the hydrogen bonding responsible for the protein conformation but will also give a net negative charge to the protein essential for its migration towards the anode during electrophoresis. FIG. 8: The above is the typical reaction by which β−Mercaptoethanol cleaves the disulphide bond in proteins • The steps in the polymerization are as follows, – TEMED catalyzes the decomposition of the persulphate ion S 2 0 2− 8 to give a free radical as, S 2 0 2− 8 + e −1 → SO4 2− + SO4 −1• – If M is the acylamide monomer then polymerization proceeds by iterating the following, R • + M → RM • RM • + M → RMM • RMM • + M → RMM • – The dissolved 0 2 mops up the free-radical. and so on...
Page 1 and 2: SDS-PAGE Electrophoresis Anirbit De
Page 3 and 4: 3 To make Staining Solution one mix
Page 5 and 6: 5 D. Making BSA dilution solution T
Page 7: • The voltage is maintained till
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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