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His tag protein purification problem


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#16 fgd2000

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Posted 01 March 2005 - 07:48 PM

Did you notice the flow rate? If the flow is very fast the protein may not combine with the resin.So we always control the flow rate in quite slow (perhaps 10ml/40min) when we add the lysis buffer to the resin,and to completely bind the protein to the Ni column we offten add them twice. B)

#17 AllenChiu

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Posted 11 April 2010 - 08:05 PM

hi, I'm 2nd year phd student working on membrane proteins and I have an msc in enzymology

I have worked with tagged proteins but never with his;

43kDa is the size of your protein including the tag? If so, did you take into account the extra amino acids when you calculated the theoretical pI ?

did you pH the buffers very carefully? do not follow Maniatis' protocols of mixing powders at some ratios, as the resulting pH can easily be 0.2-0.5 different and it DOES make quite a difference in affinity work.

did you try to bind your protein to the Ni column in the cold room? lower temperature in many cases assists binding!

did you have DTT in all your solutions your protein was in? This includes lysis buffers, dialysis buffers etc. 1mM DTT will prevent the majority of missfolding events.

Good luck

Hi,when it comes to membrane protein,how do you obtain its full length cDNA sequence?
I can't successfully PCR my interested GPCR full length..

#18 QFBDavid

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Posted 09 August 2010 - 07:23 AM

1- chech pH of your protein buffer before and after you pass your sample throught the column, sometimes pH is not ok on the column or the buffer, they MUST be the same!

2- try to activate your matrix prior to pass by your sample

3- temperature allways helps! =)




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