Hi,
I was doing protein purification today and i observed that my niNTA beads turned brown after i bound my lysate with the beads. I eluted my protein but still the color of the beads were same.
I knew this situation occurs when DTT is added since DTT reduces the nickel.
But while performing two different protein purification I faced this problem in one. Although i used the same buffer composition for both the proteins.
Why it happened in one column not in case of the other protein?
My lysis buffer composition for both the proteins was
Tris,Back,1mmDTT,pmsf,BHCL,lysosyme,glycerol,10mm immidazole
Thank you.
Hi!
Your question is why the beads turned brown in reducing conditions in one prep but not the other while using the same lysis buffer?
In the later purification your DTT might have been degraded already, it is not very stable in aqueous solutions especially at higher pH such as in the Tris buffered range.
Best,
Stefan
Hi there,
As the color turn due to DTT occurs for DTT concentration above 1mM, it might be that the concentration was not exactly the same in your 2 batches of buffer. An other explanation might be that one of the lysates contains a more reductive power than the other (due to the presence of a protein bearing numerous exposed thiols)
Hi Dipanwita,
Generally, DDT reduces the nickel and changes the color of the column. This phenomenon can also occur when the protein concentration in the lysate is high or there are many impurities.
Hi Dipanwita,
I would suggest you to dilute the lysate (preferably 1:1) before loading on the column, keep the buffer solution same (except DTT).
Also if you have not packed the NiNTA column recently, try recharging Ni-NTA beads before next purification.
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