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

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12 • The software “ImageJ” is used to ”invert” the contrast of the image and convert this inverted picture into a matrix of numbers which gets saves as a text file. FIG. 13: Inverted image of the strip of the scanned image of the gel along the dilution series • Inversion is done to make the numerical analysis more intuitive so that places in the image which are brighter because of the dye attaching to the protein have higher numerical values. • By looking at this picture the rectangular regions are selected in the strip which correspond to the protein spots and a C++ program was written which takes this text file as input, stores it as a matrix and sums the numbers in every rectangular region corresponding to each protein spot. I. The data analysis • So at the end of the data extraction one has a set of pairs of numbers {m i , I i } where I i is the intensity of of the spot corresponding to m i mass of the protein and m i+1 = 1 2 m i. Using Mathematica7 the following two graphs are plotted, – I i vs m i – I i vs i • The graph I i vs m i shows somewhat of a linear behaviour if the extreme values are dropped. At the higher mass end (i.e > 6µg) the intesity seems to stall and at the very low mass (i.e < 0.75µg) the intensity seems to drop sharply most likely because of being very close to the lowest detection limits of this staining procedure. At a protein mass of about 0.375µg the stain is just barely visible. • A typical running of a dilution gel has 6 dilutions as shown in the photographs of the strips and the intensities as defined earlier as the sum of the matrix entries in the sub-rectangles look like , {4.68083 × 10 6 , 3.7271 × 10 6 , 3.01742 × 10 6 , 2.69898 × 10 6 , 2.52403 × 10 6 , 2.40741 × 10 6 } corresponding to {12µg, 6µg, 3µg, 1.5µg, 0.75µg, 0.375µg}. This data can be analyzed in the following ways, • One can plot the points {(1, 4.68083 × 10 6 ), (2, 3.7271 × 10 6 ), (3, 3.01742 × 10 6 ), (4, 2.69898 × 10 6 ), (5, 2.52403 × 10 6 ), (6, 2.40741 × 10 6 )} and fit the function I = A x n to it and find the optimum value of A and n for that. The way to think of this graph is that given the set of points {(m i , I i )|i ∈ {1, 2, ..., 6}} one is constructing from it a set {(i, I i )|i ∈ {1, 2, ..., 6}} such that i = 1 + log 2 ( m 1 m i ). Here which is m 1 and which is m 2 etc is unambiguous since we know precisely in the making of the dilution series that m i+1 = 1 2 m i. So m 1 is always the highest mass sent through the gel.
13 FIG. 14: A pot of the points (1 + log 2 “ m1 m i ” Mathematica7 as A ∼ 4.71182 × 10 6 and n ∼ 0.385681 , I i) and fitted to it is the function I = A x n with the best fit values given by FIG. 15: A plot of the points (m iµg, I i) for the same rectangular strip from the same run with BSA as above FIG. 16: A plot of the points (m iµg, I i) for another run of a dilution gel with BSA which showed the typical problems of the intensities stalling for larger masses and showing sharp drops for lower masses. These are issues with the sensitivity of the detection technique. • For a fixed protein the above analysis is repeated on multiple gels by taking multiple rectangles around the stained strip and the values of intensities and the coeffcients A and n are averaged over them and the standard deviation of them is taken as a proxy for the error in their measurements. Displayed below are the values for the protein Actin. • Actin that was used was extracted in-house and hence the exact amount of the concentration of the protein solution is not known. But we can still do a dilution series gel using it since not knowing the exact concentration is not a hindrance in taking half of the mass inserted into the first gel ans so on. Hence for such cases we scale the mass unit and consider the highest mass lane to have “1 unit mass” Actin and the lane to the right of it has “0.5 unit mass” Actin and so forth. Then the similar image analysis can be done as explained before.
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: 11 H. Image analysis on the gel Aft
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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