HELP! This is my first attempt to immunoprecipitate insulin receptor beta using protein A sepharose. My initial attempts using the IP kit from Upstate have all failed. I ordered Protein A Sepharose from Amersham but now I dont know what to do with it. Does anyone have a detailed protocol on how to use it? It is in a solution of ethanol, but I assume this has to be washed. And what does it mean to "preclear" my lysate?

Any help would be greatly appreciated!!

ac said:

HELP! This is my first attempt to immunoprecipitate insulin receptor beta using protein A sepharose. My initial attempts using the IP kit from Upstate have all failed. I ordered Protein A Sepharose from Amersham but now I dont know what to do with it. Does anyone have a detailed protocol on how to use it? It is in a solution of ethanol, but I assume this has to be washed. And what does it mean to "preclear" my lysate? Any help would be greatly appreciated!!

This an immunoprecipitation protocol for Protein G sepharose from Amesham. Protein A must have similar protocol. It explains how to preclear your lysate. To make sure you are doing the right thing, contact Amersham using this URL:

http://www.amersham.com/contact.html


Immunoprecipitation

Materials:
primary antibody:
approx 2-5 ug of antibody

protein extract:
prepared in relatively physiological buffer

Protease Inhibitors:
1:100 protease inhibitor cocktail

Protein G Sepharose:
Amersham (17-0618-01)

Wash Buffer:
Extract buffer or other physiological buffer (increasing salt concentrations may be necessary to increase stringency of immunoprecipitation)

2X Sample Buffer:

Procedure:
Prepare Protein G Sepharose
Add 100 ul of sepharose slurry (=50 ul bed volume) per immunoprecipitation to eppendorf
Wash sepharose 3x with extract buffer
Spin down beads 30 sec @ 600 x g (2000 rpm in IEC clinical centrifuge)
Remove supernatant above bed of sepharose
Resuspend sepharose in 1 volume buffer to make 50% slurry
Preclear Extract
Add fresh protease inhibitors to approx 5-8 ug of total cell extract
Add 50 ul Protein-G Sepharose slurry (=25 ul bed volume) to extract
Rotate @ 4 degrees for 1 hour
Spin out Protein-G Sepharose
Transfer supernatant (=precleared extract for step 3) to new tube
Wash sepharose beads 3x with wash buffer or extract buffer
Add 25 ul of 2X sample buffer to sepharose and boil 10' (=proteins pulled out by Protein-G Sepharose alone)
Immunoprecipitate antigen of interest
Add 2-5 ug of precipitating antibody to precleared extract
Rotate @ 4 degrees for 1 - 3 hours
Precipitate antibody-antigen complexes by adding 50ul (25 ul bed volume) of Protein-G Sepharose slurry
Rotate @ 4 degrees for 30 min - 1 hour
Spin down Protein-G - antibody - antigen complexes
Wash 4 x with extract buffer or wash buffer
Add 25 ul of 2X Sample buffer and boil for 10' to release complexes from Protein G Sepharose
Process for Immunoblotting


Notes:
Amounts of extract and antibody used are starting approximations. Experiments should be adjusted for the relative abundance of the protein of interest.
Protein G Sepharose can be used to effectively precipitate complexes from either mouse or rabbit antibodies
Very important to do control immunoprecipitations to show specificity which include:
no antibody but Protein-G Sepharose (i.e. from the preclearing step)
non-specific antibody or normal rabbit serum
When western blotting, it is useful to probe with a different antibody that recognizes the same antigen than was used for the immunoprecipitation steps to show specificity
If different species antibodies are available, use a different type for the western than the immunoprecipitation - otherwise you will also detect the Ig heavy chains from the immunoprecipitation
Can eliminate the Ig heavy chain problem by first crosslinking the immunoprecipitating antibody to the Protein G-Sepharose - that way only the antigen (not the antibody) will be released from the Protein-G Sepharose following boiling in Sample buffer.
References:
Protein Methods, D.M. Bollag et al. (1996)
Short Protocols in Molecular Biology, Ausubel et al, (1998)
Antibodies, a Laboratory Manual, E. Harlow & D. Lane, (1988)

After such a detailed protocol I just can salute you.
I have some minor things to think about.
1- wash the g-bead at least 3 times in 1 ml of the buffer.(getting rid of the ethanol is very important).
2- choose the correct buffer for your protein.
3- add not just protease inhibtor coctail. add also 1nMPMSF and 10mM Iodoacetamide, if you see degradation of your protein.
3- Sometimed boiling the beads + the elution buffer isnt good, you can also try incubating them for 20min at 37.
Otherwise
Good Luck

pre-clearing means you add beads to your lysate and spin it out to get rid of any non-specific binding. you do this before adding the antibody.

Hi..

How does boiling of SDS liberate the antigent from the protein A?

Boiling in SDS sample buffer completely denatures the Protein A, the antibody and the antigen. This prevents any interaction of the antibody and antigen. Unfortunately, it also means that you'll be seeing a huge amount of antibody heavy and light chains on your gel/western.

I wouldn't use this method if you're interested in proteins that migrate in the 50-60 kD range or 25-35 kD range because they will be obscured by the large amount of antibody. To look at proteins in these size ranges you need to covalently attach the antibody to the agarose resin. Pierce sells several kits designed for this, or you can use their instructions and try to do it yourself.

Here's one of their options that also comes with a couple options for preclearing and other controls. I used this kit to precipitate chaperone/heat shock proteins and their binding partners:
http://www.piercenet.com/Products/Browse.cfm?fldID=CE353C79-DB1C-4501-B8E2-FA96342112DC