I have a custom antibody (it works for Western blot and immunohistochemistry) that I would like to try for immunoprecipitation. I know that the antibody successfully binds my beads (I'm crosslinking the antibody to the resin prior to mixing them with my tissue lysate). What controls are necessary? (all to be assessed by Western blot)
I would definitely have the following:
positive control: load a lane on your gel with a given amount of purified recombinant protein of interest (your target), this whould give you a single defined clean band which you could use as a reference
- you should definitely included aliquots of all the supernatants, washes and flow throughs to make sure the immunoprecipitation is specific for however you are eluting and that your target is not being lost in any other fractions.
-I'd also conduct a reaction using lysate from cells where the protein is either knock-down or not expressed (is your protein endogenous or epitop tagged in any way? This could be a useful control for specificity to show your antibody only selects the protein of interest)
-of course you could also so a small titration using increasing amounts of protein IP lysate just in case the protein is expressed in low yield, this would allow you to get an idea for its limit of detection in terms of total protein collect from a lysate after pull-down.
- one last useful control would be to load a lane where the beads has been denatured to release the antibodies, that way you can detect bands that might be coming from the light and heavy chains of the primary antibody (this also will ensure that your antibody has efficiently crosslinked to the beads, or maybe you've done this already?)
hope this helps!
You should have, as in each experiment, a positive and a negative control.
That means:
Find a protein which is known to bind to the protein of interest (the protein you have an antibody against) and analyze it with western blot, if this protein can be detected.
You also should show that the IP worked -> test if the protein which you used the antibody against is detectable also on the western.
Also use beads without antibody binding or bound with a non specific antibody -> you should not see any bands on the control western.
If you know a protein that for sure does not interact with the protein of interest you could also use this as a negative control.
I hope that helps.
Hi Sebastian,
That helped a lot, thanks.
I'm a bit limited in the world of positive controls: I do not know what other protein(s) my protein of interest binds to, nor do I have any antibodies I know should bind the resin (I work with sea urchins, so we don't have commercially available antibodies....). For the Western blot part, I'd definitely include total lysate for my Western blot (I'd be doing this prior to IP as well to make sure my protein is in fact present).
In terms of other controls, so then
1) beads+antibody post-IP (actually see my proteins if the IP worked)
2) beads mixed with lysate without any antibody present as a negative control to see what is non-specifically binding the beads
3) would it be a good idea to run the beads+antibody alone to ensure that the bands I see are actually protein and not just antibody? (I expect to pull down a family of related proteins of varying molecular weight for which my antibody is selective for a conserved region)
4) I figure I should also run the flow thru of each step as a control of sorts.
Does this sound sufficient? Having never tried this with an antibody not designed for IP, I'm quite nervous about leaving out any prudent controls....
Thanks!
Lauren
hey,
I think that sounds good.
What I am doing usually is to add the antibody directly to the lysates rotate over night at 4°C and on the next day I just add Protein G magnetic beads (without any antibody bound) for 3 hours to the mixture of lysate and antibody.
within this 3 hours the protein g beads should bind the antibody and after this time I just wash my beads and should just have proteins which are bound to antibody-beads.
Maybe this why of doing would also be ok for you?
I often do IPs with antibodies which are not tested for IP. But with the correct controls also that is possible.
all the best,
Sebastian
Interesting; I've always seen this method as magic, but worth a try.
Do you add any cross-linked to cross-link the antibody to the beads, or just mix them together? Would it be possible to use a cross-linker using that method? I'm hoping to use the proteins in functional studies in the future, so am using the a crosslinking kit (with A/G beads) so that I can (hopefully) elute the proteins without the antibody.
Thanks again,
Lauren
well I just add the beads to the mixture.
and I elute with sample buffer plus 95°C for 10 minutes.
I cannot tell you if you can also use a crosslinker, but I would not do it if you add the beads to the antibody/lysate mixture, because it could lead to wrong interactions.
Each method is like magic, we always have to show that the magic is true ;)
best,
Sebastian
I would definitely have the following:
positive control: load a lane on your gel with a given amount of purified recombinant protein of interest (your target), this whould give you a single defined clean band which you could use as a reference
- you should definitely included aliquots of all the supernatants, washes and flow throughs to make sure the immunoprecipitation is specific for however you are eluting and that your target is not being lost in any other fractions.
-I'd also conduct a reaction using lysate from cells where the protein is either knock-down or not expressed (is your protein endogenous or epitop tagged in any way? This could be a useful control for specificity to show your antibody only selects the protein of interest)
-of course you could also so a small titration using increasing amounts of protein IP lysate just in case the protein is expressed in low yield, this would allow you to get an idea for its limit of detection in terms of total protein collect from a lysate after pull-down.
- one last useful control would be to load a lane where the beads has been denatured to release the antibodies, that way you can detect bands that might be coming from the light and heavy chains of the primary antibody (this also will ensure that your antibody has efficiently crosslinked to the beads, or maybe you've done this already?)
hope this helps!
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