The Cation Exchange Gel Samples

1175 WordsApr 24, 20175 Pages
The cation exchange gel samples are labelled IEX 19-22 as these were the tubes with the highest activity as shown in the above table. The other IEX fractions had significantly lower specific activities so were less likely to contain the protein of interest and more likely to contain unwanted proteins than the higher specific activity fractions. Only the higher specific activity fractions were chosen for this reason. All the fractions that were analysed by gel electrophoresis have the same banding patterns which means that they can be combined to get a combined total for the yield calculations. This has then been used to calculate the amount that there would have been if the entire solution was used for this process. However, the…show more content…
This is seen in the gel electrophoresis where there is a greater number of bands for dye 9-11 than there is for the IEX tubes. The fold purification was expected to be between 3-7-fold as found on the “BRENDA web page” (2017), for this experiment a 1.4-fold (492404/349710) increase in activity was seen after dye ligand chromatography. The retrospective yield was like cation exchange which also gave an impossible result considering the after-dialysis yield. It is difficult to say which yield is more accurate. The after-dialysis yield measured may be lower than its true value or the error created by averaging the different tubes and treating them as if the entire sample has overestimated the amount of LDH present. Although there are more bands on the gel there is a lower retrospective total protein for dye ligand than there is for cation exchange 43.2 mg and 55.9 mg respectively. This could indicate that although there is a greater number of bands the ratio of LDH to unwanted proteins may be higher. This technique has gotten rid of the band that was present in cation exchange but has different bands present which shows that the two purification techniques are targeting LDH in different ways. Affinity chromatography relies on the protein ability to bind to specific molecules tightly but not covalently. This technique uses a ligand bound to the matrix that is capable of specifically binding to the protein. When the impure solution is passed through the
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