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Chris McDermott-Roe
Aug 11, 2012, 6:29:44 PM8/11/12
to met...@magpie.bio.indiana.edu
Hi,
I’m having some IP issues and I’m (seriously) hoping somebody can
help. Basically,
I’m successfully pulling down my protein BUT it’s also present in my IgG IP.
I’m guessing my washing conditions aren’t stringent enough but I’m slightly
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!!
I’m having some IP issues and I’m (seriously) hoping somebody can
help. Basically,
I’m successfully pulling down my protein BUT it’s also present in my IgG IP.
I’m guessing my washing conditions aren’t stringent enough but I’m slightly
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!!
Irit Rappley
Aug 11, 2012, 10:41:11 PM8/11/12
to Chris McDermott-Roe, met...@magpie.bio.indiana.edu
Hi Chris,
Sounds like you just need to optimize your conditions.
1. What is the concentration of your lysate? It should be no more than about 1 mg/mL.
2. Do you pre-clear? Your protein (and lots of others) could be sticking to the beads themselves. Incubate your samples for ~1 hr at 4C with unconjugated beads to pre-clear. You can keep these beads, elute the proteins off them, and see what is coming down.
3. If you still need more stringent wash conditions, you can try RIPA buffer and/or play with the salt concentration. For example: wash 1X PBS + Tween, then 1X high salt (500 mM), then 1X RIPA. You could also add a final detergent-free wash, which further helps to get rid of non-specific binding. Often it's not so much about the specific conditions as about changing them with each wash.
Hope this helps,
Irit
________________________________________
From: methods...@oat.bio.indiana.edu [methods...@oat.bio.indiana.edu] On Behalf Of Chris McDermott-Roe [cjmcder...@gmail.com]
Sent: Saturday, August 11, 2012 3:29 PM
To: met...@magpie.bio.indiana.edu
Subject: (no subject)
_______________________________________________
Methods mailing list
Met...@net.bio.net
http://www.bio.net/biomail/listinfo/methods
Sounds like you just need to optimize your conditions.
1. What is the concentration of your lysate? It should be no more than about 1 mg/mL.
2. Do you pre-clear? Your protein (and lots of others) could be sticking to the beads themselves. Incubate your samples for ~1 hr at 4C with unconjugated beads to pre-clear. You can keep these beads, elute the proteins off them, and see what is coming down.
3. If you still need more stringent wash conditions, you can try RIPA buffer and/or play with the salt concentration. For example: wash 1X PBS + Tween, then 1X high salt (500 mM), then 1X RIPA. You could also add a final detergent-free wash, which further helps to get rid of non-specific binding. Often it's not so much about the specific conditions as about changing them with each wash.
Hope this helps,
Irit
________________________________________
From: methods...@oat.bio.indiana.edu [methods...@oat.bio.indiana.edu] On Behalf Of Chris McDermott-Roe [cjmcder...@gmail.com]
Sent: Saturday, August 11, 2012 3:29 PM
To: met...@magpie.bio.indiana.edu
Subject: (no subject)
Methods mailing list
Met...@net.bio.net
http://www.bio.net/biomail/listinfo/methods
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