His-tagged protein purification problem
Posted 01 June 2011 - 01:40 AM
Posted 01 June 2011 - 10:16 PM
Hola before digestion you have to be sure of have a quasi pure protein preparation , improving Ni-colum elution (see WB forum today answers)or making a second purification step as gel filtration or ion exchanger. and check the purity of your band after any step before digestion. Buena suerte
I am trying to purify rainbow trout recombinant myoglobin. The coding region of rainbow trout myoglobin was successfully inserted into pET-32a(+) vector. The His-tagged (N-terminal) fusion protein was over expressed in BL21(DE3)pLysS competent cells, successfully harvested, sonicated and affinity purified by Histrap HP Columns (molecular weight of the recombinant protein is around 36kDa [20 kDa from vector + ~16 kDa from rainbow trout myoglobin]). I encountered problems when I tried to cleave the His-tag by enterokinase (Enterokinase was from Roche,Calf intestine). After checking the cleavage extent by SDS-PAGE, I found several bands around the expected position of my target protein. So, I performed N-terminal amino acid sequencing to confirm the corresponding myoglobin band. But the N-terminal sequencing revealed that the bands were from thioredoxin. How can I improve the cleavage condition? Any suggestions or recommendations?
Posted 27 June 2011 - 10:12 PM
i am sammer, a new forum member.
i am facing the difficulty to purify my HIs-tag protein. its size is 19KD. the problmis after purification i found two prominen bands one is around 60KD and other is 19 KD. also i want to know that after NI-NTA step, can i load my protien on GEl filteration column directly without precipating with ammonium sulfate.