For metal ion chromatography I am using equilibration buffer, wash buffer and elution buffer for elution of protein and further absorbance reading at 280 nm. Blank set with elution buffer gave negative protein absorbance, however, setting blank with equilibration buffer protein fraction showed positive results. Can anyone suggest why this might have happened? There is only a difference of sodium chloride presence in equilibration buffer and not in elution buffer.
Dear Shalini
I gather that you may be using using a step wise elution of your His-tag (or like) protein from a Ni or like column using imidazole (or like) in your eluting buffer. Because the elution buffer composition in the fractions is changing over die chromatographic run, it may be difficult to choose the most appropriate blank. To explain ...First, during the elution, you column are not fully equilibrated during the initial phase (first few fractions). Therefore the buffer composition in these fractions may differ (lower concentration) than the elution buffer that you prepared, as some of the buffer components (ie imidazole for Ni-based chromatography) will associate with the column matrix. If you measure the "blank/buffer" sample and your fractions after blanking against an empty dry cuvette, you will see will see that the absorbance is lower than that of the elution buffer. You will, however, see your early eluting peak fractions if you plot the data. Second, If you protein elutes in the later fractions better equilibration will have taken place, but the buffer composition will still be a little different in the fraction, and particularly in your protein fraction which will contain less competing buffer compound, for example imidazole. We often see a drifting baseline for such type of chromatographic runs, as imidazole and such compounds have UV absorption.
My advise it to use either an empty cuvette or your equilibration buffer to blank the spectrophotomer. You do not need absolute absorbance values to draw a graph of your chromatograhy. If you really want to try an cancel the drift and get closer to the absolute 280 nm absorption values, you will need to do two identical runs with the same buffer and chromatography parameters, preferably on the same day with control run without protein and the experimental run with protein. Measure the correlating fractions and subtract the controls from the experimental fractions. Plot it and see if it works. However, I do not think is it worth spending too much time on such endeavor if you find your protein containing fractions with the more simple analyses.
Good luck!Regards
marina
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts