Ask help for protein purification using His-tag column!
khatipovNO-SPAM at NO-SPAMuchicago.edu
Tue Oct 22 12:01:18 EST 2002
Do you treat your samples with DNAse? Do you see any turbidity when you do
same manipulations with the extracts of cells not expressing your
recombinant protein? If yes, then it is most possibly DNA that makes you
samples dense. Try DNAse-treating before loading. However, be careful if you
protein has DNA-binding properties, because digesting the DNA can
destabilize it. If no, then it is your protein that aggregates and clogs the
column. It happens when one overexpresses the protein and most of it does
not fold correctly. If turbidity results in low yields, I would recommend
doing chromatography under denaturing conditions and then learning by trial
and error how to refold the protein.
<chengzhjun at hotmail.com> wrote in message
news:20021022055838.24989.qmail at ww02.hostica.com...
> my protein is about 30kDa and its pI is 5.1. I used pH=8.0 PBS binding
buffer to banlance the Ni-His tag column. but when i put the protein
solution into the column,the protein solution becomes turbid and the resin
aggregates together and so the protein buffer could't flow down. How can I
do with it .
> Any information would be appreciated!
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