Asper discussion, First time my clonning was failed becuase the first 45 nucleotide was not cloned in pet30 a vector then I choosed the different clone of 1:1 ration fraction, I got the size of purified protein near to the expected size 17.7kda and very thick band but there was also thin nonspecfic protein band saw in Western blot result whose size is more than 20kda but less than 23 kda but size of band is very thin. can anyone please tell me ? what could be the reason ?
It is the riboflavin kinase protein of leishmania donovani, i used this protein only as control for compare the size of control protein and expected size of our protein.
because in last clone after induction and expression, I got less than 15kda but in this clone size of protein interest is some how equal to the size of expected protein
I purifying it by using Ni-NTA methods
i was asking about LdRFK because it has exactly the same size as your "contaminant". The contaminant is somehow connected to your protein of interest, because the amounts of both always vary together. which expression system are you using? is it E. coli? I am asking because it could be protein glycosylation, which would only be present in eukaryotic expression systems.
yes sir, it is pET 30a E.coli Bl21 (de3) cells
the cloned gene from A. baumannii so most of the strain of A. buamannii have glycoylation properties.
So what should we do for resolving this problem ? by which we can get only 17.7 kda protein.
I have no idea, but option (1) is that your protein is getting modified in some way in E. coli. You need access to mass spectrometry to address this. It could (2) be a co-purifying protein (such as a chaperone). Again, this can be identified by mass spec. Have you tried purification under denaturing conditions (in urea)? Is the faint band also there? Option (3) is to do a second purification step using anion or cation exchange materials.
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