| | 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.