Immunoprecipitation - (Aug/06/2009 )

Hi, do you guys have some good protocol for IP ?

What i was trying to do is using an antibody to trap the target protein. The following is what i did.

1) Incubate the antibody with the cell lysate
2) remove the unbind cell lysate
3) Add Protein A and incubate
4) Elution

The question that i have , is that i am having a hard time in separating my antibody and my target protein
in the elution fraction, it contains my antibody and also my target protein.

help ....

thanks

sorry, it's a really brief description of your protocol and I don't get it. You incubate your cell lysate with antibody and then you remove your unbound cell lysate? How can you do that? is your antibody already bond on beads? I don't get your protocol. Give more details if you want some help.

What I do is:
incubate the sample with antibody
incubate the sample + antibody + protein A coupled to beads
wash
elute by boiling with loading buffer

I get my protein and the antibody used for the IP into the gel.
If I don't want to see the antibody while performing the western blot, I don't use an HRP-anti-IgG, but I rather use HRP-protein A to detect the primary antibody. This will not bind to denatured immunoglobulin used for IP

IP is tricky, but it's not hard....

you just need two primary antibodies raised in different species if your problem is how to detect the protein in the western blot part.

for example, IP with ab raised in mouse
and then wb with same ab but raised in rabbit, followed by using secondary ab against rabbit.

Curtis

Curtis on Aug 6 2009, 12:03 PM said:

not working with all antibodies. I had already some cross-reaction between mice and rabbit antibodies.
I was very happy with HRP- protein A

little mouse on Aug 6 2009, 05:47 PM said:

Will the boiling denatured my target protein ?

i found so many protocol online, they all said that after the elution, boil the sample to the SDS page or Western Blot to analysis the result.

But is there a way to separate the target protein and the antibody ?
Because i have to use the pure product to test for antiviral effect...

Thanks

Danny Chow on Aug 7 2009, 05:41 AM said:

I see, you are purifying the protein.
you have to use a column with covalently bond antibodies. Don't look for IP protocols, it's not the same purpose (in these protocols they use few amount and load all what is IPed on a SDS-PAGE. You would have to look for immunoaffinity purification.
I don't have my lab book here right now. ... I'll try to find a protocol.

Depending on your antigen-antibody interaction, you might try different elution conditions.
Have a look here

http://www.piercenet.com/Objects/View.cfm?...BA-8DFBC1B802D3

And here you would find some information about the agarose :

http://www.piercenet.com/products/browse.cfm?fldID=01020201

(I was using CnBr agarose.)

little mouse on Aug 7 2009, 03:09 PM said:

I tried this before, but it failed to capture anything... plus... i am expressing this protein in mammalian cell ... the yield is very low.. the only method for detecting this protein is western blot... so troublesome ..
But anyways, thank you for the advice !
i think i will try it one more time by using this immunoaffinity chromotography