methods to remove Ig chains from immunopreciptation elution

[ahubbard]

I want to co-ippt two endogenous proteins. I want to avoid having Ig heavy and light chains in my final eluted sample. How do I best remove them, short of purifying them and cross linking them ala Pierce kit to beads?

[kevin_a]

What is the reason you want to remove the IgG from the sample? Only so they don't show up on a Western?

Give me some details and I'll be glad to outline a protocol for you.

[ahubbard]

Kevin,

I want to identify the interacting protein by coomassie or silver stain. the regions of 25 kD and 50kD are common protein sizes in the lliver, where I am working. ANN

[kevin_a]

Ann,

Keep in mind that crosslinking the IgG to the resin is going to be more robust. You don't nessesarily have to purify the IgG to do this. All you need to do is add the IgG to the the Protein A (or G) beads and then crosslink. This doesn't always work well with monoclonal antibodies, but when it does you can often reuse the columns a few times.

That said...
Since you are doing a gel stain, you can't ignore all the other proteins that will be present in the samples like would be possible in a Western. So, in this case, the simplest way to solve your problem is to just salt-off the interacting partner(s) and leave the bait attached to the column-along with the Antibody. A high salt elution will leave the IgG attached to (I assume Protein A). Depending on the affinity of your antibody, you might be able to release the bait protein off the antibody with some SDS.

So for elutions, try...
1) Just Prey Proteins: add 200mM-700mM NaCl to your wash buffer and use as an elution buffer (may or may not work depending on the strength of the bait-prey interactions.

2) Prey and Bait: add 200mM-700mM NaCl to your wash buffer along with 0.05-0.5 SDS. (may or may not work depending on the strengh of the antibody. RIPA buffer is commonly used for IPs, but some antibodies just won't function in that much detergent. If yours is like this, you might be able to recover quite a lot of bait protein with a little SDS and leave the IgG behind.) At the higher SDS concentrations, you might see the IgG start to come off and I can't recall if ever found a threshold for it.

Hope this helps.

[kiki06]

Good suggestions... from what I remember too there are specific beats that you can buy (or other similar kits) that bind to your IgG (that you use to IP) and either let you purify it or weigh it down so much that it either never enters the gel or makes it easy to centrifuge it away from your sample after immunprecipitation.

let us know how your IP goes

[marcroboy]

Hi everyone, I'm in a similar situation as ahubbard. I'm immunoprecipitating a 40kd transcription factor with the use of Dynabead Protein G.

I have done my cross-linking reaction however it did not appear to be working. I am pretty positive that the subsequent western blot did not detect my protein, only the antibody that was eluted. I have done an elution before adding the proteins to collect any un-linked antibody. I'm worried that my cross-linking wasn't strong enough that I eluted most of the antibody before the immunoprecipitation. I have used citrate buffer pH 2.88 for all my elutions.

So I have two questions here: Is there a easy way instead of running the whole IP and western experiment to check that my antibody were crossed linked properly? Maybe by comparing the concentration of the eluted antibody between crossed-linked and uncross-linked Protein G-antibody complex?

And also is my elution buffer too strong? It was suggested by the manual to use citrate buffer pH 2-3 but I don't think my antibody binds very well to Protein G. Can anyone suggest a gentler elution buffer and pH?

Thanks a bunch

[kevin_a]

Marcroboy -

The acid elution is your problem with the Dyna Beads. They Dyna Beads, Magna Bind Beads, et al are silanized to put a reactive surface on the iron core and then the capture protein is attached via the coated reative surface. The Amino Silane (typically) used to coat the beads will elute off in acid... Thus you have Antibody and Protein G coming off along with your protein.

If you crosslinked the antibody to the Protein G - Try a basic (pH 11) elution buffer such as ethanolamine or maybe even just Urea. Agarose beads are going to be a much better choice for that.

However, since you are doing a Western Blot - you could likely just leave everything alone and use the Thermo Scientific Clean Blot Reagent. It's a HRP conjugate that only binds to native IgG. So the denatured IgG and Protein G will not show up on your Western. It's cross species reactive so it doesn't really matter much what your primary antibody is.

[marcroboy]

Originally Posted by kevin_a

Marcroboy -

The acid elution is your problem with the Dyna Beads. They Dyna Beads, Magna Bind Beads, et al are silanized to put a reactive surface on the iron core and then the capture protein is attached via the coated reative surface. The Amino Silane (typically) used to coat the beads will elute off in acid... Thus you have Antibody and Protein G coming off along with your protein.

If you crosslinked the antibody to the Protein G - Try a basic (pH 11) elution buffer such as ethanolamine or maybe even just Urea. Agarose beads are going to be a much better choice for that.

However, since you are doing a Western Blot - you could likely just leave everything alone and use the Thermo Scientific Clean Blot Reagent. It's a HRP conjugate that only binds to native IgG. So the denatured IgG and Protein G will not show up on your Western. It's cross species reactive so it doesn't really matter much what your primary antibody is.

Interesting, the pH 3 elution buffer was suggested by the product manual (only mixed for 2 minutes), it would seem strange for them to put it there if it would elute off protein G from the beads. Do you have an reliable source where I can confirm this information?

Thanks for the answer btw, appreciate it.

[kevin_a]

Not sure why they recommend a pH 3. We suggest people avoid doing that because of the leaching. Your incubation time is short, so that may prevent significant leaching. Then again my intel could be wrong about the chemistry on the Dyna beads, but I do trust the source.

If you just want to check the crosslinking efficiency: Before you do an actual experiment, perform a Western on the washes and pre-elutionion fractions with anti-IgG after you do the cross-linking so you can see what's coming off and when. If you see IgG chains light up on the Western at 25, 50, 75 and 150 then - the crosslinking is not working. The recombinant Protein G is ~22 kDa, so bands additional bands at 22, 47, 72, 97, 172 would indicate the Protein G came off with the IgG.


There is also another possible explanation for why you don't see your protein of interest. The elution conditions may be too mild. More common than you might thins, some IgG will bind very tightly to antigen and then resist standard elution conditions. Your protein is 40 so you should be able to resolve that from the mess that will result by simply boiling in reducing SDS sample buffer after the IP and doing the Western.

If you need more detailed help, feel free to keep posting or email me.

K

[marcroboy]

Originally Posted by kevin_a

Not sure why they recommend a pH 3. We suggest people avoid doing that because of the leaching. Your incubation time is short, so that may prevent significant leaching. Then again my intel could be wrong about the chemistry on the Dyna beads, but I do trust the source.

If you just want to check the crosslinking efficiency: Before you do an actual experiment, perform a Western on the washes and pre-elutionion fractions with anti-IgG after you do the cross-linking so you can see what's coming off and when. If you see IgG chains light up on the Western at 25, 50, 75 and 150 then - the crosslinking is not working. The recombinant Protein G is ~22 kDa, so bands additional bands at 22, 47, 72, 97, 172 would indicate the Protein G came off with the IgG.


There is also another possible explanation for why you don't see your protein of interest. The elution conditions may be too mild. More common than you might thins, some IgG will bind very tightly to antigen and then resist standard elution conditions. Your protein is 40 so you should be able to resolve that from the mess that will result by simply boiling in reducing SDS sample buffer after the IP and doing the Western.

If you need more detailed help, feel free to keep posting or email me.

K

Hi Kevin,
I think I have the answer to my failed immunoprecipitation: as it turned out, my purified protein sample was degraded, probably because of the lack of protease inhibitor. So yea, no wonder I was having problems, I was IPing out of nothing

However, I have run an assortment of elutions under different conditions to test my cross-linking efficiency. I hope with your familiarity with this technique you could give me some pointers.

I ran an IP without cross-linking then I eluted with 0.1 M citrate pH 3 buffer. As expected I see the heavy chain and light chain band under comassie (distinctly). Furthermore, with the same bead,I also boiled them under reducing loading buffer after the first elution, I could also see the antibody band(faintly). And this band is slightly higher than the acid eluted band.

On a different set of beads, I ran the IP with cross-linking. First elution with citrate pH 3 buffer gave me a faint heavy chain band. The second acid elution gave me nothing.
Consequently, I boiled the same beads under reducing loading buffer and again it gave me the heavy chain band. And just as the uncross-linked case, this band is slightly heavier than the acid eluted band.

I guess the second case would suggest that crosslinking worked? I didn't get antibody with the second acid wash however boiling them under reducing condition resulted in the antibodies.

Furthermore, it seemed that every time I boil the beads, the resulting band is always a bit higher than the acid eluting the beads. However, I can't imagine what else would be coming off besides the antibody and protein G. What are the chances that protein G are coming off with boiling?

Thank you