Non-specific binding of polyclonal antibodies - help (Nov/21/2007 )
hi all..
I have raised antibodies in rabbit against a purified protein. When i did western with the antiserum i could see another non specific band at higher molecular weight other than the original one. I also have a mutant organism which does not make this protein. Can anyone suggest me how to preincubate antibody in a protein mix from the mutant and then use it for western.
Thank you
-transposon9-
QUOTE (transposon9 @ Nov 21 2007, 01:33 AM)
hi all..
I have raised antibodies in rabbit against a purified protein. When i did western with the antiserum i could see another non specific band at higher molecular weight other than the original one. I also have a mutant organism which does not make this protein. Can anyone suggest me how to preincubate antibody in a protein mix from the mutant and then use it for western.
Thank you
I have raised antibodies in rabbit against a purified protein. When i did western with the antiserum i could see another non specific band at higher molecular weight other than the original one. I also have a mutant organism which does not make this protein. Can anyone suggest me how to preincubate antibody in a protein mix from the mutant and then use it for western.
Thank you
Non-specific bands are common when making antibodies, and they may not pose a problem so long as you can easily detect your specific band and convince others that it is specific. Since you have a mutant (lucky you!!), I would go ahead and try to do a western without any purification to start with. You might get lucky and have very little clean up to do.
-smu2-