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Co-Ip of Membran Protein Troubleshoot

Co-Ip Lysis Buffer Membraneprotein

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#1 biopace

biopace

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Posted 05 April 2012 - 02:36 AM

Hello,

I´m fairly new to this forum, but I already gained insight in the problems involving Immunoprecipitation through various existing posts. So thanks already in advance for your help.

I performed the Co-IP already several times without any luck. Now I repeated the IP with similar proteins where a successfully co-ip was already performed. Again my ip was negative.
Protein A is a transmembran protein (HA-tagged). Protein B is a membrane associated protein. So maybe my lysis buffer is crucial and not ideal?

The protocol I use:

Day 1: Prepare HEK cells on a 6 well plate
Day 2: Transfection with Fugene and 2500ng DNA for each Protein.
Day 3: Pbs Wash. Lysis with Lysis buffer (150mM NaCl, 50mM Tris ph7,4, 1mM EDTA, 1% Triton + PMSF + Phospatase Inhibitor Cocktail). Shock-Freeze in liquid nitrogen and after rethawing mechanical lysis with a syringe 26G. Centrifugation for 15 min at max speed (14000rpm). collecting the supernatant.
Co-Ip: adding HA Ab (rabbit) and incubate at 4C over night
Day 4: adding A-Sepharose Beads for 4 h at 4C .Centrifuge 1min at 13000rpm. wash one time with Puffer i used for lysis. Then I add Laemmli Puffer and boil for 10 min at 70C. Centrifuge to collect the supernatant and add Mercaptoethanol + DTT to the samples and boil once more at 70C for 10 min.
Run the Gel and blot on PVDF and use Protein B Ab (mouse).

I can't see the specific band of protein B. Ab contamination isn't likely as Protein B is at 80 kDA My controls are plain lysate and is positive for protein B and Protein A.
I also have high background noise and unspesific bands in the Ip.
Tried precleearing once with another Ab but it wasn't much of an improvement.

Any suggestions on my protocol or buffer. I really appreciate any help, as this is rather frustrating.
thanks




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Edited by biopace, 05 April 2012 - 02:42 AM.






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