(no subject)

Chris McDermott-Roe via methods%40net.bio.net (by cjmcdermottroe from gmail.com)
Sat Aug 11 17:29:44 EST 2012


Hi,



I=92m having some IP issues and I=92m (seriously) hoping somebody can
help.  Basically,
I=92m successfully pulling down my protein BUT it=92s also present in my Ig=
G IP.
I=92m guessing my washing conditions aren=92t stringent enough but I=92m sl=
ightly
worried that this non-specific binding is occurring in the first place.  Is
this normal?





My protocol is pretty simple, it's as follows..

1.      Prepare a heart protein lysate using Cell Signaling lysis buffer
(0.1% triton plus usual buffers).

2.      Incubate 300 micrograms of protein lysate with Dynabeads (50
microliters as recommended by Invitrogen) already bound to an antibody (2
micrograms total) against my protein of interest overnight.

3.      Wash beads three times with PBS (+0.02% tween).

4.      Re-suspend beads in 50 microliters laemli loading buffer and run
gel/western


Thanks!!


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