IP for few hours or ON - what's your favorite method? (May/28/2006 )
Hi
I want to do an IP.
I am considering trying a new and quicker method. But I would like to have your opinion on it.
Quick method:
- incubation of the rabbit polyclonal antibody (Ab) with protein G beads for ~ 1hour.
- wash beads with lysis buffer to wash away non-bound Ab
- incubate protein lysate to Ab-beads "conjugate" for 1 hour
- wash
-add sample loading buffer and run on SDS-PAGe gel.
the advantages of this protocol are:
- is quick, everything is done in one day
- less protein degradation
-less background
Until now I have always done my IPs in the following way:
- pre-clear lysate with protein G beads, 30min
- incubate lysate with Ab ON 4C
- incubate lysate/Ab complexes with protein G beads, 3 hours 4C
- wash
-add sample loading buffer and load on gel
What's your favorite IP method and why?
I would like to try the quick method because of the advantages I have mentioned above.
Have you ever tried such quick IP method?
Usually I do like your previous method, but only 1 hour with the protein G, and I preincubate the beads with BSA before to add the cell extract (I absolutely don't want any background)
why, because I never thested the quick way. It seems good, unless for the background, i'm sceptical? I never tried. I'm interested.
Tell us.
Hello-
I do a lot of IP's and I usually just add the antibodies 1st for like 5 mins while I go find the protein G beads and then add them too and rock at 4C for 1.5-3 hours. I've heard some antibodies need a longer incubation to IP efficiently but so far with the antibodies I have been using 2 hours works fine.
In terms of background reduction I have come across two tricks. Covalently coupling your antibodies to the beads eliminates those annoying heavy chain bands. Also someone recently told me to try drastically limiting the amount of sepharose beads you use, and it does seem to work quite nicely to reduce background. (I tried only 5ul for a bazillion cells last week and stained my IP gel with colloidal coomassie and see much less bands in the IgG control lane)
[quote name='Mountainman' date='May 29 2006, 11:25 PM' post='53434']
Hello-
I do a lot of IP's and I usually just add the antibodies 1st for like 5 mins while I go find the protein G beads and then add them too and rock at 4C for 1.5-3 hours. I've heard some antibodies need a longer incubation to IP efficiently but so far with the antibodies I have been using 2 hours works fine.
Hi Mountainman,
You do incubate everything (antibody, protein G beads and protein extract) more or less at the same time? Hum... I must say that way I never heard.
But you do wash the G beads 3 x with lysis buffer before adding to protein extract, don't you?
Which kind of antibody are you using? Polyclonal, Monoclonal? Is it puified antibody?
Thanks
To avoid to see the heavy chain of the Ig that was used to IP, you can detect the Ig used for WB with protein A-HRP. works fine. it doesn't recognize the denatured antobodies.
you can also use protein G-HRP, but it slightly recognize the denatured Ig. So you should reduce the amount of beads to reduce the background.
Yeah, in the process of changing buffers multiple times for the covalent coupling the G beads get washed probably more than 3 times actually. I've never really worried about antibodies that aren't bound to the beads because well they should be removed in all your final washes for the immunoprecipitation.
The antibodies I've been using are mostly monoclonals, but I'm about to try it with a purified polyclonal tomorrow. I can only assume it should work too.
you can also use protein G-HRP, but it slightly recognize the denatured Ig. So you should reduce the amount of beads to reduce the background.
Hi everybody!!!
Missele could you tell me what Protein A-HRP do you use, I mean the company. I often had a problem with heavy and light chain, and would like to try Protein A-HRP.
Thank you in advance,
Lucised
To avoid to see the heavy chain of the Ig that was used to IP, you can detect the Ig used for WB with protein A-HRP. works fine. it doesn't recognize the denatured antobodies.
you can also use protein G-HRP, but it slightly recognize the denatured Ig. So you should reduce the amount of beads to reduce the background.
Hi everybody!!!
Missele could you tell me what Protein A-HRP do you use, I mean the company. I often had a problem with heavy and light chain, and would like to try Protein A-HRP.
Thank you in advance,
Lucised
I use a protein G-HRP , from molecular probes P21041, but it detects slightly the light chain.
The protein A-HRP doesn't detect it. I think it was also from molecular probes, but I can't find again the reference.
Protein G is better for mouse IgG1, and rat Ig.
Missele, many thanks for your kind reply!!!
Usually, what I do to avoid seeing heavy and light chains is that I first use polyclonal and than monoclonal antibody. but of course I can not completely get rid of them.
I will try Protein A- and G-HRP.
Lucised
you're welcome Lucised.
Actually, after further investigation, the protein G-HRP doesn't detect the light chain of Ig, but it something else, slightly bigger. something that is precipitated with the protein g-agarose beads. It's really dependent on the beads. I changed the provider, and I had no more this band.
So, the protein G-HRP should be really OK.