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IP-Western Problems


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#1 anonymous

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Posted 09 November 2001 - 10:00 PM

Hi

I'm having problems with immunoprecipitation followed by westernblot experiments. If I immunoprecipitate with a mouse Ab and blot with a rabbit Ab the secondary anti-rabbit-HRP antibody picks up the mouse Ig on the membrane. Same problem if I IP with a rabbit Ab and blot with a mouse Ab - the anti-mouse-HRP picks up the rabbit Ig on the membrane. And wouldn't you know it, the protein I'm interested in is about 50 kDa. I am using my immunoprecipitating Abs at 1:5000 dilution (monoclonal culture supenatants or rabbit serum) and the secondary-HRP conjugated Abs at 1:10,000.I have tried secondary HRP conjugated Abs from various companies with no luck.

Any suggestions?

CheersFiona


#2 anonymous

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Posted 19 November 2001 - 10:00 PM

Dear Fiona !

just try to use 'precleared' secondary antibodies i.e. a secondary anti rabbit ab pretreated with mouse serum to deplete all ab that crossreact with precipitated mouse ab.

ab like this are available from many companies i.e. Santa Cruz ....etc.

Daniel


#3 anonymous

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Posted 27 November 2001 - 10:00 PM

You might try looking at the HRP-labeled ProA conjugates available at Zymed Laboratories, Inc. It has a weak affinity for mouse Ig and strong binding with rabbit Ig. Maybe with some creative dilution, you could make it work for you.

#4 hbassett

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Posted 15 February 2002 - 02:51 PM

I also recommend pre-clearing.  Also, if you run your sample under non-reducing conditions (ie no DTT or BME) then your Igg will run as one mass (not heavy and light chain) at ~150 kd.  I do IP's for a protein at ~53kd and this helps.

#5 labslave

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Posted 21 November 2002 - 10:13 AM

You can try to covalently link your primary antibody to CNBr beads and run your IP through that. Also I agree with hbassett by not adding any reducing agents to your loading buffer before you run your western would also help.

#6 BioGuru

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Posted 15 August 2004 - 10:30 AM

Hi,

Have you tried using TrueBlot for your IP Western? It is designed specifically for this purpose - to reduce hinderance of heavy and light Ig chains.

for anti-rabbit IgG and anti-mouse IgG detection:

http://www.ebioscien..._18/18-8877.htm

http://www.ebioscien..._88/88-1688.htm


for more info, e-mail me at tech@ebioscience.com

Good luck!
:P




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