Why does the initial protein elution (elution No 2)(Each elution volume 500microL) in maltose binding protein purification exhibit high concentrations of the target protein on SDS-PAGE, but fail to demonstrate high activity compared to the lateral elution fractions with lower target protein concentrations? Can the activity of the target protein be disrupted by the expression of other proteins related to the vector (PMALC2X)?
Are you sure that the big band you see in elution No 2 is the desired protein? Have you checked by sequencing or by mass spectrometry?
Since you did not say what the protein in question is, it is not possible to speculate on whether its activity could be inhibited by another protein expressed from the same plasmid. It would also be helpful to say what other proteins are encoded by this plasmid. Nevertheless, it seems unlikely.
How are you measuring the activity of your protein?
Adam B Shapiro Thank you sir for providing information. I have verified the target protein by sequencing. However, it is intriguing that the lateral elution (No 3-6) are showing the activity, but the elution no 2 with big band; there is no activity. The protein has glycosidic bond cleavage ability. To assess the activity, I have conducted the DNS method.
Is it possible that elution no 2 is protein that did not stick to the column because it is aggregated, whereas the later fractions are protein that bound to the column? Aggregated column might not be active because it is not in its native conformation.
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