5 replies to this topic

[eukaryote]

Dear Friends, Please help me solve this strange problem quickly
I used Pet 30 a vector and Rosetta 2 de3 cells. I expressed 2 proteins, and one of them should be 22 kda but it is showing 29 kda and another one should be 17 but its like 23 kda on SDS-PAGE. I added his tag and other tags inframe when expressed. When I used nickel column, the protein bound well and all the fractions showed the same increased size and eluted well. Has any one experienced this problem. If you please let me know how you solved your problem, that woud be very helpful. I look forward to hear your words of wisdom as soon as possible.
Thank you

[swanny]

eukaryote, on Oct 18 2009, 04:37 AM, said:

Dear Friends, Please help me solve this strange problem quickly
I used Pet 30 a vector and Rosetta 2 de3 cells. I expressed 2 proteins, and one of them should be 22 kda but it is showing 29 kda and another one should be 17 but its like 23 kda on SDS-PAGE. I added his tag and other tags inframe when expressed. When I used nickel column, the protein bound well and all the fractions showed the same increased size and eluted well. Has any one experienced this problem. If you please let me know how you solved your problem, that woud be very helpful. I look forward to hear your words of wisdom as soon as possible.
Thank you

What RE site(s) did you use? Could you have removed a stop codon?

[lna]

Did you always have the increased size of the products with this vector, or it occured after some manipulations? Because sometimes you can get the translation from the alternative start-codon, which is not even an AUG.. in this case you can only try to make the shift in the reading frame, which corresponds to the amternative initiation point. Of course, if you have the His-tag in the N-terminus, it could not be the case. In This case there could be a loss of stop-codon. So, check the sequence?

[swanny]

Also, is there anything strange about the amino acid sequence? Sometimes proteins will run at a different apparent mass.

[Prep!]

swanny, on Oct 26 2009, 08:11 AM, said:

Also, is there anything strange about the amino acid sequence? Sometimes proteins will run at a different apparent mass.

Also post transationally modified proteins like glycosylated ones show a higher molecular weight in gels due its hydrodynamic radius!!!

[Prep!]

Also check for the mol wt any any other orthogonal technique just o be sure!!!