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  2. → Viewing Profile: Posts: Bioscientist

Bioscientist

Member Since 20 Jul 2011
Offline Last Active May 27 2013 10:22 PM
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Posts I've Made

In Topic: Trouble with purified recombinant protein bands

24 May 2013 - 09:01 AM

Thanks for the reply.

The only difference is that the upper band has a score of 2216.42 and the lower band has a score of  2082.55. Do you think this slightly truncated version is due to proteases? I thought that if due to protease I would get a lot more smaller peptides in the SDS-PAGE gel. And what are the post-translational modifications possible in E.Coli?

In Topic: trouble in his-tagged protein binding with Ni-NTA agarose

02 May 2013 - 10:42 AM

No I am not trying to wash it. I am trying to elute using 250mM in the first case and 300mM imidazole in the 2nd case.

Even after using 300mM imidazole, I still find my proteins attached to the agarose. I am asking if due to the binding buffer this happens?

In Topic: Protein purification problems: Presence of contaminated proteins along with the

18 January 2013 - 01:48 PM

Thanks a lot for the replies. I was wondering if I use size exclusion chromatography what media or resin should I use for the chromatography?

In Topic: Western blot trouble shooting

29 August 2012 - 12:40 PM

Thanks Andreea.

My protein is about 29 KDa and I transfer for 1 hour at 25 volts. I can reduce it to 30 minutes and see what happens.

And my samples doesn't look viscous compared to the control. I will decrease my loading amount of sample.

And I was also wondering if I cut the gel and extract my protein from the gel and use that to run western blot, do you think it can reduce the smearing effect?

Thanks.

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