Problem in western blotting

rs_santhosh at hotmail.com rs_santhosh at hotmail.com
Sat Jun 29 00:07:33 EST 2002


I raised antibody against a his-tagged protein that was overexpressed using pQE vector.I used difference in pH for eluting the protein from the ni affinity column.I was able to see the protein only after the affinity purification.A single band was eluted at pH 4.5.That protein band I used for raising antibody.In dot blot with purified antigen I got good signal.But when I did western blot It is showing lot of affinity to two host protein bands(crude cell lysate).And my desired band is not lighting up but signal is there with purified band(control).Used PVDF for blotting and used PBS as buffer for incubation and washing.What is your suggestions to see my protein band in crude host lysate 

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