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how to get pure protein - (Sep/20/2010 )

I want to purify 55 kda HIs tagged pro of very high purity from bacterial expression system. please guide me how to proceed. I have done NI NTA first and then I want to do ION Exchange. BUTI am having problems with IN exchange.I am not able to standardise it.And after purificaion how should I check the purity of my protein


Hola, IMAC is enought reproducible, at least mantaining buffer concentrations and volumes as a FPLC do; only you could appreciate a lost of capacity of column if any of the buffer components affect to it, but you can see a lost of colour of resin. To do an ion exchager after it, you have to decrease the imidazole concentration by dyalisys or better diluting tenfold your positive fractions pool. To see the quality of your protein peak made a PAGE, and to quantify compare the intensity of your band with known samples of BSA wich has a similar MW. Apply 1,3,and 5ug in some wells and in the same gel one,two or more wells with your pure sample. Good Luck