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-   -   Protien alterations causing band shift? (http://www.molecularstation.com/forum/immunoprecipitation-forum/4052-protien-alterations-causing-band-shift.html)

MWRLAB 10-02-2008 02:30 PM

Protien alterations causing band shift?
 
I'm working with a protein 68 KDa in size. I immunoprecipitated and probed my membrane with the same antibody. After probing my PVDF membrane I get a strong signal ~75 KDa in the input lane, where as I get a signal in the IP'd lanes right at 68KDa. Is there some kind of modification that could account for this shift? Or is there another explanation?

frederic6342 02-04-2009 10:17 PM

Re: Protien alterations causing band shift? similar problem
 
Hi, I have a similar problem.
I've performed an immunoprecipitation targeting a protein of 65-70 kDa in a tissue lysate using a rabbit polyclonal Ab and protein A-Agarose conjugated.
After washing, I've suspended beads in reducing agent and LDS buffer from invitrogen as recommended for a classical WB, treeating them 10 minutes at 70°C. Samples were stored at -80°C until electrophoresis.
Then, I performed protein electrophoresis using NuPAGEŽ Novex 4-12% BisTris Gel, Transfer, blocking, incubation with the same Ab followed by incubation with Anti rabbit 2nd Ac-HRp conjugated. Revelation using Ecl revealed a band at 50KDa (Heavy Ab chain as oftenly found) and a unexpected band at approx 90kDa is detected., nothing around 70KD. Is it possible that something remains bound to my protein (Ab light chain..) or what should it be (my unprecipitated lysate shows a band at 70KDa thats seems that my protein was present?
Is it enough to incubate samples at 70°C instead of 100°C as frequently reported. Should I better use sample treated in Laemmly buffer on NuPage Gels?

Thank you in advance.


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