high backgroud on western - (Sep/08/2006 )

hey guys!

i'm still having troubles with my western blot.
i seem to have a high background.
how can i solve this problem in a fast way?
i happen to wash my membrane for 5 mins for 3 times
with ample amount of PBS/0.1%Tween.
i am handling 6 proteins for western.
somethimes i get high backgrounds and somethimes i don't.

please help...

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do some antibodies always give high backgrounds and others don't? - try asking the companies that supplied them for advice

how long are you blocking for??? you could try blocking overnight.

What is your primary antibody diltuted in? try it in 5% milk

You could also try washing the membrane in 5% milk in TBST after the secondary antibody

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I've had the same problems. Here are some suggestions:
1) Try primary antibody in 5% milk in TBST or 5% BSA in TBST
2) Try blocking overnight (5% milk) rather than primary Ab
3) Blocking 1hr at 37C and then 1hr at rommtemp
4) I have added washes - 3 quick ones and 5 of 5min
5) Using a lower concentration of the secondary antibody, even if it increases exposure time

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