IP-Western Problems - (Nov/09/2001 )
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.
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.
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.
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.
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.
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:
for more info, e-mail me at firstname.lastname@example.org
Santa Cruz has developed a system to eliminate heavy and light chain in the exact situation you are describing.
If you are using a mouse for the IP and a rabbit for the WB, the ExactaCruz Kit C: sc-45450 would be most suitable to eliminate the heavy chain and only detect your protein of interest.
If you are using the rabbit for the IP and the mouse for the WB, then ExactaCruz Kit B: sc-45039 would be most suitable.
If you are interested in getting more information, you can either call us at 1-800-457-3801 ext. 2 for technical service, or go to the What's New link on our website www.scbt.com and click on ExactaCruz.