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Dave_Kub_11's Content

There have been 2 items by Dave_Kub_11 (Search limited from 11-August 19)

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#165534 Precipitation during coupling

Posted by Dave_Kub_11 on 26 February 2014 - 06:47 AM in Protein and Proteomics

Checked the pH after the precipitate forms and it is between pH 8-8.5, which I guess it should be as the coupling buffer is pH 8.3. 


The coupling buffer is 0.1M NaHCO3, 0.5M NaCl pH 8.3


The protein has been dialysed and stored in this buffer and when I add the protein to the beads, I top up with some buffer to ensure the beads are incubated in a decent volume.  Its during this step the precipitation occurs so I don't understand how adjusting the pH would help?  (sorry)

#165479 Precipitation during coupling

Posted by Dave_Kub_11 on 25 February 2014 - 08:20 AM in Protein and Proteomics

Hi there,


I have been having an issue when coupling my GST/His-tagged protein to CnBr sepharose beads.  When I add my protein to the coupling buffer and the CnBr beads, an egg-white-like precipitate forms after a few minutes of mixing (which I assume is the protein precipitating out!).


This is strange as the protein I am using has been dialysed in the same coupling buffer with which I use to couple the protein to the beads. I have already troubleshooted this extensively and it still can't explain this!


The first time I did this, the precipitation occurred but I carried on with the coupling and funnily enough it appeared to have worked as I was able to pull down proteins by western blot with the coupled beads! 


Even so,  I am still perplexed as to what could cause this and am worried if the efficiency of the coupling is being lost.  I would appreciate any ideas!


(The first time I did this, I used 2.5mg protein with 0.5ml bed volume of CnBr sepharose beads.  Since then, I scaled up using 5mg to 1ml bed volume beads).



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