His tag protein purification problem
Posted 01 March 2005 - 07:48 PM
Posted 11 April 2010 - 08:05 PM
Hi,when it comes to membrane protein,how do you obtain its full length cDNA sequence?
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.
I can't successfully PCR my interested GPCR full length..
Posted 09 August 2010 - 07:23 AM
2- try to activate your matrix prior to pass by your sample
3- temperature allways helps! =)