Hi Debajyoti,
There is another simple thing you can try: Raise your NaCl concentration
to 500 or 1000 mM in your lysis buffer. This helps to clean up your
sample further and it might inhibit proteases in your lysate.
Good luck,
christian
Debajyoti Dutta wrote:
Hi,
This is going to be an off topic question concerning this community. I
have a protein 6XHis tagged. When retrieved from the Ni-NTA column with
imidazole found to be degraded, appears like a deep band with other
bands (touching each other below the main band) in SDS PAGE. The protein
is a DNA binding (pI~ 10) and Tris-Hcl buffer is used with 10% glycerol
and 300mM NaCl. for purification. Does Phosphate buffer do any help in
stopping the degradation.
All suggestions are welcome. Thank you for your reply in advance.
Sincerely
Debajyoti Dutta
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_______________________________________________________________________
Dr. Christian Biertümpfel
Laboratory of Molecular Biology
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Bethesda, MD 20892-0580
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