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

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4 FIG. 2: The above photograph shows green frames in which the gel is set and the transparent rack in which the green frames are clamped to keep them stable during the polymerization process. • Now one mixes the APS and the TEMED in the stacking solution and pours it into the gap between the pairs of plates through a pipette in a similar fashion. Now one has to quickly insert the comb into the gap which will demarcate the wells that will form inside the stacking gel. This comb is not to be taken out untill the protein loading starts. • This set up should be left undisturbed till it is necessary to install the gels into the electrophoresis tank. • Generally during this time one starts making the chemicals that will be loaded into the gel. Like the dilution series of Actin and BSA to be described below. C. Making Actin dilution solution When Actin is extracted it is not known as to how much Actin is present in the solution. It is estimated from indirect methods that the concentration is 2µg/mul. The various steps in preparing the Actin dilution are as follows, • I took 5 aliquots of the Actin solution which gave me about 43µl of Actin solution. • I added 117µl of H 2 O to it and hence made up the solution to 160µl. Call this solution A 0 • I took 80µl of A 0 and added 80µl of H 2 O to it and made 160µl of another solution. Lets name it A 1 • Again I took 80µl of A 1 and added 80µl of H 2 O to it and made 160 of another solution. Lets name it A 2 . • Thus iterating the process about 5 times one makes solutions of 80µl each till A 5 . In the making of A 5 the 80µl of A 4 that remains is preserved separately for future experiments and not used in the dyeing and heating in the next steps. • In each of the 80µl solutions from A 0 to A 5 of Actin, 20µl of the “5X” DTT dye is added (so that in the final solution the dye is in concentration of “1X”) and then each of these 100µl solutions is sent for heating for 5mins at 100 ◦ C . • Post heating one loads 20µl of each solution in each of the wells of the gel. Hence the amounts of protein in the wells of the gel fall in inverse powers of two.
5 D. Making BSA dilution solution The steps to prepare the BSA sample that will be loaded in the gel, 1. 5mg of BSA is mixed in 1ml of water. (call this solution A 0 ) 2. 0.5ml of A 0 is taken in another aliquot and 0.5ml of water is mixed in it (call it solution A 1 ) 3. 0.5ml of A 1 is taken in another aliquot and 0.5mk of water is mixed in it (call it solution A 2 ). 4. In this way solutions till A 5 are made. 5. 30µl of A 0 is taken in an aliquot and 7.5µl of the ‘5X” dye is mixed in it. (This choice is explained in the last point) 6. So the aliquout now contains 150µg of BSA in a solution of volume 37.5µl. 7. To denature the proteins boil this 37.5µl of solution for 5min at 100 ◦ C (call this B 0 ) 8. The steps 5,6 and 7 are repeated for A 1 , A 2 , A 3 , A 4 , A 5 and call the corresponding 37.5µl solutions B 1 , B 2 , B 3 , B 4 , B 5 . 9. 3µl of each B i is taken and loaded into each of the wells. 10. Then the mass of the BSA loaded in each of the wells are respectively 12µg, 6µg, 3µg, 1.5µg, 0.75µg and 0.375µg. 11. The ′ 5X” dye is a solution of the dye made and stored in such a way that it has concentration 5 times more than what it should be in the final solution. Then one can see that adding 7.5µl of such a ‘5X” dye to 30µl of fluids results in the correct concentration of the dye in the final mixture. The steps are as follows, E. Loading, running, staining and destaining the gel • One generally needs to have about 500ml of the electrophoresis buffer as a conductor for running the gel. • 50ml of the available 10X buffer is mixed with 450ml of autoclaved water to make the required solution. • First the two glass framed gels are slid into the slots on the two sides of the frame that goes into the tank. The glass frames are positioned so that the shorter plate faces inward. • Checking for snug fitting one locks them by the plastic hinges on the frame. • Then the frame is inserted into the tank and the electrophoresis buffer is poured into the volume between the two glass frames. • One checks if the buffer is leaking out into the tank. If it does then one has to diamantle the frame and reset it again. • Then one pours the rest of the prepared buffer in the volume surrounding the frame inside the tank. • Now one should pull out the combs taking care that the jerk does not break the wells inside the gel that would have formed. • At certain angles the wells will be visibile to the naked eye because of the refractive index difference between air and the transparent gel. • To reduce chances of loading the protein at regions other than the gel one can mark the positions of the wells on the wall of the tank using some marker. • The proteins are loaded into the micropipettes from the aliquots and the pippete is lightly inserted into the well from the side of the shorter glass plate and the protein pushed in. One can see the coloured protein settled into the well. Care should be taken to push in the protein in one shot since doing a suction can contaminate the protein by the buffer solution. • After all the gels are loaded, the top cover is closed and it is connected to the D.C source that comes with the instrument. It is generally run at about 100V . This D.C source has ratings of delivering 2.5A current at 500V delivering a maximum power of 500W .
Page 1 and 2: SDS-PAGE Electrophoresis Anirbit De
Page 3: 3 To make Staining Solution one mix
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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