After purifying my recombination fusion protein sample using a Bio-rad Q-column chromatography, the resultant samples, were verified via SDS-PAGE analysis and stored in glycerol 20% concentration (0.201 mg/mL) and digested using 0.5uL of Factor Xa, incubating overnight at 4 degrees. The factor Xa cleavage result was positive. However pure CCL5 protein was not retrieved after apply 20uL of MBP sahparose to 180uL of digested fusion protein after an incubation period of 2hours
And further purified using a Bio-rad Q-column chromatography, the resultant samples, were verified via SDS-PAGE analysis and stored in glycerol 20% concentration (0.201 mg/mL) and digested using 0.5uL of Factor Xa, incubating overnight at 4 degrees. The factor Xa cleavage result was positive. However pure CCL5 protein could not be retrieved from the supernatant, after applying 20uL of MBP sahparose to 180uL of digested fusion protein after an incubation period of 2hours and centrifugating at high speed.
what can i go differently please?
You could try to separate the CCL5 and MBP on the q column after the Factor Xa digestion, instead of using the MBP-sepharose (amylose resin?). The isoelectric points of CCL5 and MBP are 9.27 and 5.41, respectively, so they should be easy to resolve on the q column.
MBP affinity chromatography is the best option. Even after cleavage the sample can be applied to the resin and cleaved protein can be retrieved from the flowthorough.
Good luck
Hi Wisdom
This paper contain the method using affinity chromatography
Good Luck
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