9E10 - myc - Western Blotting - HELP!

Peter French p.french at ARNIE.CFI.UNSW.EDU.AU
Thu Sep 14 00:59:30 EST 1995


>Does anyone know of special comditions for immunblot detection of the 
>c-myc epitope using 9E10 monoclonal antibody.
>
>Currently I transfer my PAGE gel to nitrocellulose,
>block with 5% skim milk powder in PBS,
>probe with 9E10 supernatant,
>wash with PBS/Tween
>probe with antimouse-HRPO in 5% skim milk powder in PBS,
>wash with PBS/Tween, then detect with ECL.
>
>However, I am getting many non-specific bands coming up, and I thought 
>that 9E10 was quite specific for myc.
>
>Does anyone know of a way to elminate these background bands?
>
>Thanks in advance,
>
>Andrew (Melbourne, Australia).


Dear Andrew,
The problem may be with your secondary antibody conjugate. Try either
diluting it more (eg 1/1000 or greater depending on brand) or try a
different brand of secondary antibody. Some give more non-specific binding
than others. One more thing, try diluting your solutions in PBS containing
0.5% skim milk powder rather than 5%. Good luck,


Peter French, Centre for Immunology, St Vincent's Hospital, Sydney





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