problem in immunoprecipitation - (Nov/30/2006 )

Hi to everybody who can help me with my problem in immunoprecipitation!

I'm using a rabbit polyclonal anti-GHSR1a antibody to immunoprecipitate GHS-R1a from lysates which surely express the receptor. I'm using Protein A-Sepharose (Amersham) with 6 microg of antibody and 1 mg of lysate. At the beginning I used to incubate the antibody with Protein A for 2 hours and then to add the precleared lysate and incubate it O/N.
I've also tried to incubate first the precleared lysate and the antibody O/N and then to add the Protein A for 2 hours but nothing has changed.
The horrible thing is that my protein seems to come out of the IP, in fact I find it in the supernatant that I usually store before I begin to wash the beds of the IP...

Can anyone help me?

Thanks a lot...

Cris

---

Hi,

I'm not exactly sure what your problem is because the following sentence you
wrote was ambiguos;
"The horrible thing is that my protein seems to come out of the IP, in fact I find it in the supernatant that I usually store before I begin to wash the beds of the IP..."

Are you saying that your antibody does not deplete the protein from the supernatant? This is unclear.

Suggestions.
#1) Protein G beads work better than protein A in my hands regardless of isotype.
#2) has it ever been shown that the antibody you are using can actually immunoprecipitate
your protein of interest? Some antibodies work for Western but not for IP.

---

Hi Mike,

thanks for your reply!

At the moment there are only 2 commercial anti-GHSR1a antibodies that work well for western. First I used the Alpha Diagnostic antibody but it didn't work well in western neither in IP. Now I'm using the Phoenix rabbit polyclonal anti-GHSR1a and it works well in Western but it has never been tested for IP by Phoenix. I've read an article where the authors use this antibody to immunoprecipitate GHS-R1a from cell lysate (Kim MS et al, Mol Endocrinol. 2004 Sep;18(9):2291-301). I'm sure this is the only one published article about IP with Phoenix anti-GHSR1a antibody.

I know that I'm trying to use an antibody that perhaps doesn't work well for IP but I haven't a lot of experience in IP so I'd like to have the opinion of people that are more confident with this kind of detection...

I use the same antibody to detect the immunoprecipitated protein in WB and what I can see are only heavy and light chains. Well, I see also other bands of weak intensity but the pattern is exactly the same as in the negative control (the membrane is incubated only with the secondary rabbit antibody).
I can see my protein only in the lysate and in the supernatant that I usually store after IP (before the washings)...

---

is there any antibody for ELISA?
it would recognize the native form of your antigen for sure.

---

I've just checked it in the data sheet of my antibody and here it's written that "the HPLC purified standard peptide, GHSR1a, can be detected with this IgG in ELISA"...

Cris

---

Hi again,

Ahhh, now I see what you mean. You IP your protein, bind the antibody with sepharose beads
and then save the supernatant as a control. Well, it sounds like your experiment is very well
controlled, you do a negative with IgG (or beads alone) and check your supernatant for immunodepleteion.
I have a few suggestions that may (or may not) help.
#1 (you may be doing #1 already) When you do a Western for your IP, load an input from your original protein sample (~10%, using a defined amount of protein) and also load the same amount of protein from your immunodepleted sample. ie. if you load 50 micrograms of protein from your original protein sample, also load 50 micrograms of protein from the sample that you have IPed.
By doing this, you can compare the total amount of protein in 50 micrograms (or whatever amt.) or your original sample versus 50 micrograms of your IPed sample and thus will be able to tell if you have removed any of your protein during the Immunoprecipitation.
#2 Use protein G sepharose
#3 Your protocol sounds good but maybe you could try my approach.For my IPs I IP with my antibody overnight at 4 degrees (always) followed by antibody capture with protein G for 4 hours (slow process but works well).
#4 in the event that you are unable to get the Phoenix antibodies to work, clone the cDNA encoding your protein into a FLAG, or HA, or GFP tagged vector, transfect into your cells and then IP using anantibody against the tag.

Good luck!!!

---

PS

Since you currently are not pulling down your protein, make sure you try using a low stringency IP and wash buffer.
For example, use a buffer with no more than 0.1% detergent and no higher than 50 mM NaCl.
Was no more than 3 times with this same buffer. If the antibody-antigen interaction is weak, the antibody may not bind under harsher conditions such as when using RIPA.

---

I agree with Mikew,
but I would like to add just one comment : your antibody recognizes the peptide by ELISA. It doesn't mean it would recognize the native protein.

---

Hello Mike,

Thank you very much for your reply, I'll try to apply some of your suggestions to my protocol!

Cris

---