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

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16 FIG. 19: The above plot shows the fall of the intensity with 1+ logarithm of the fracional mass along with an estimate of the errors in the intensities and the curve I = 2.95005×106 is fitted to that. x 0.197972 FIG. 20: The above graph shows the intensities of the Coomassie stains as a function of the mass of the protein in micrograms in the BSA sample along with an estimate of the errors in the intensities. This shows the problems of stalling at higher masses and sharp drop of the sensitivity at the lower ends. It also shows the best-fit straight line of 56709.4490m + 2.2349 × 10 6 that one gets on dropping the lowest mass from the data. J. Using the results of the analysis If the largest mass of the protein used in the dilution gel is m 0 (with respect to which the other masses are scaled) then the result of the above calculation states that there exists constants A and n such that the intensity (I) of a stain goes with mass of the protein (m) as, A I = [ ( m0 ) ] n log2 m + 1 But one must be cautious of the interpretation of this result as it stands now. One should take note that the constants A and n are NOT intrinsic to the protein but depend on m 0 as can be easily checked by doing the fit on the same set of data with the current highest mass removed and scaling everything with respect to the current second highest mass. But obviously for the same m 0 , A and n will be different for different proteins.
But for a fixed protein if m 0 and its corresponding A and n are known then one can treat the result as a standardization such that it can now be used to predict masses of other samples of the same protein from their staining intensities. But the prediction can have two different forms as follows, • Like for standardization with BSA where m 0 is known, given an unknown sample of stain intensity I its mass can be predicted to be m = 2 m 0 “ ” 1 A n I −1 1 • Like for standardization with Actin where m 0 is not known, given two samples of proteins of stain intensities I 1 and I 2 one can predict the ratio of the masses of the proteins as, 17 m 1 m 2 = 2 “ A I 1 ” 1 n − “ ” 1 A n I 2 K. Some findings/observations of my own • I started filtering the destaining solution twice to ensure greater efficieny of the chemical on reuse. • I devised this method of transferring the gel on the scanner by using plastic supports while keeping it always submeged in the destaining solution. This practically eliminates the risk of air bubbles getting trapped. • The image analysis technique described here is of my own and I have not found that in any reference. • I wondered if the staining/destaining step could be avoided by sending into the gel, protein mixed with Coomassie Blue. But then this does not work as I found out that Coomassie Blue happens to have higher mobility than the proteins and hence inside the gel it moves faster ahead without causing any staining effect. III. SOME THEORETICAL BACKGROUND The entire physics and chemistry of the process of electrophoresis is a very complicated mix of phenomenon and much of the numbers like the amount of each chemical to be used is very hard to rationalize by pure theoretical calculation and are obtained by experimental optimization. A. A basic overview of the idea One of the key ideas behind electrophoresis is that if 2 solutions of different pH and different densities containing charged molecules of different mobility can be stabilized against convective instabilities then the boundary between the two fluids can be robustly maintained in an electric field. In that case the two fluids can move maintaining the boundary or if the boundary is somehow made rigid (like through polymerization) then the charged ions can flow through it in a predictable sequence without disturbing the boundary. This predictability of the sequence gives the quite essential “stacking” effect in gel electrophoresis which holds the key to identification of the different protein components in the initial solution. The convective instabilities are generally avoided by either having the two fluids to be immiscible and of different densities or by polymerization like here so that convection is prohibited simply due to structural rigidity. If the boundary is moveable then the crucial observation is that inspite of the difference in mobilities of the mobile ions on the two sides the velocities at which they move have to be the same since otherwise a gap gets created at the interface through which electricity cannot conduct. Since in most situations the electric field and the mobilities are given constants the formation of the gap is prevented by the rigid polymer matrix, the system attains this dynamic quilibrium by dissociating the “correct” number of ions on either side. Eventually it wll be seen that whether the system can dissociate to this optimal extent depends on whether the experimentalist has chosen the right dissociation reactions and pH. And much of these optimal values have been established over years of standardization of the experiment and only some order of magnitude esimates can be gotten from pure theoretical arguments.
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 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: 15 FIG. 18: The above graph shows t
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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