Jackson Immuno Research has Light and Heavy chain specific secondary antibodies. So you would use the LC specific secondary to detect your protein around 55KD (you will not detect any heavy chain), and the HC specific for the lower molecular weight protein. (you will detect no light chain)I have had good luck with it.

Ya I can give u a hint what I tried. I am sure you do heating at more than 90 degrees with a denaturing agent(DTT or ß-ME) to elute your binding protein(s) on the beads(am I right?).What I can suggest is, do the same heating step without a denaturing agent, collect the supernatant(hopefully your protein only!), then make a second heating step with your favourite DTT or 2ME.

It may help.

Cheers,

AA

I have also met with the same problem very often (With IP and sometimes if u blot tissue lysates from immune cell rich tissues with antibodies of the same species).

Classic solution for the problem is using secondary antibodies raised specifically against non-denatured antibodies. I have tried the Cleanblot IP reagent from Thermo with limited succes. It was OK but if the signal is weak its tuff because atleast in my hands its very weak compared to our normal secondary antibodies. Better option is conformation specific secondary antibody from Cell Signaling (but it is not labeled).

There is a Nucleic acids research paper (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1350844/) about using protein A / G-HRP instead of secondary antibodies.

(A more cheap alternative would be the suggestion from Ananda above, but I think it is applicable only if u use bead conjugated antibodies for IP.)

Good Luck

Manoj

Sorry the paper link above is not working. Its from Mol Cell Probes not NAR.

Clean western blot signals from immunoprecipitated samples

Ashish Lal,a* Susan R. Haynes,b and Myriam Gorospea

Mol Cell Probes. 2005 December; 19(6): 385–388.

:Manoj

Dear Amit,

I agree with Ananda.But you don't need to separate the sample anymore form the beads. And from my experience, unless your protein of interest contains a LOT of cysteins, even without reducing agent in the sample preparation, your antibody should recognize your protein (I have even better bands when I don't use mercapto in y sampls for about 80% of my protein tested). By not adding reducing agent your antibody bands should appear at ~150 and ~75 kDa, so your protein should not be masked anymore.

The other option: Bind your antibody covalently to magnetic beads. The protocol does not take more than 2,5 hours and the costs are within acceptable areas. I bind my purified antibody to Chemicell beads (Carboxyl-) using the CMC in their protocol (it is much cheaper than EDC and is as good) to activate the beads and then add the antibody. With 10 mg of protein you have about 200 µl of beads and for one IP, I use only 10 µl for a good band of my wanted protein. You can even reuse the beads after the pull down by separating your protein using a low pH solution. Check out their website. The advantage is that your antibody will get stuck to the beads and only your protein will be in solution. So no antibody will be in your loaded sample. Good luck!

Try TrueBlot from ebioscience as a secondary antibody for your Western. It preferentially detects native IgG, and thus you should not see the denatured IP antibody.

I think H & L are for heavy and light chains. I will think (and I am just guessing here!) that the purification takes place by affinity to the heavy and light chain of the antibodies. I have work with secondary antibodies that identify only heavy or only light chains but usually they detect both chains. I suppose it is helpful to know if both chains are detected if you need to separate by papain digestion your heavy chain from the light one, or if you have hybridoma-cells antibodies. I have an Ab that has light chain of mouse but heavy of rat... really BAD for purification, but excellent for digestion... These are only ideas, but search in internet did not make me smarter =)

Dear Amit. Another option is that you use a biotinylated primary antibody against your antigen (or you biotinylated it yourself with a simple kit) - and you use streptavidin-HRP (or ABC: avidin-biotin-peroxidase complex), which binds to the biotin-label of the primary antibody (but not to the heavy and light chain of the antibody used for IP). Good luck, Johannes

Dear Johannes, why would biotinylation of the antibody block the antigens detected by the secondary antibody in the western blot? Is it not too dangerous to do heavy biotinylation for the antibody function?.. I think I am missing a piece of the puzzle... =)

Dear Luisa. I would not biotinylate the antibody that is used for the immunoprecipitation (of protein 1) - but the antibody that is used for the detection of protein 2 (in the Western Blot). In this case both antibodies can be of the same species (rabbit polyclonal) - as you are not using an anti-rabbit IgG-HRP to detect the primary antibody in the Western Blot - but a Strepatvidin-HRP conjugate: This should not bind to the heavy or light chain of the precipitating antibody (against protein 1) but only to the biotin-moietiy of the antibody against protein 2. Of course heavy biotinylation might result in loss of antibody-antigen binding but the commercial kits are usually leading to a moderate biotinylation (resulting in just a few biotin-labels per IgG molecule). Is the concept clearer now?

Another approach that we used a lot was the use of precipitating antibodies that are covalently linked to the beads (we usually purchased flag-affinity matrix from Sigma - containing anti-flag antibodies chemically coupled to Sepharose - in case that our protein 1 was flag-tagged). When the IP-beads are boiled in SDS-sample buffer (containing SDS and DTT or ME), the heavy chain usually still sticks to the beads and doesn't get to the extract that is loaded to the gel (but only the light chain, because most of the IgG is bound via the heavy chain to the beads). Theoretically you could also bind your own specific antibody against protein 1 (not flag-tagged) to Sepharose using chemically activated Sepharose (e.g. CNBr-activated). Using antibodies covalently linked to the beads usually reduces the problem with the heavy chain significantly - but you still might significant amounts of light chain on the Western (because the light chain is washed off with the SDS/DTT sample buffer). Cheers, Johannes

Ah!!!! Thanks Johannes!, Yap! I do the covalent binding as well... I just did not understand your first proposal! Cheers, Luisa

Someone still uses to performe IP after methabolic labelling with 35S-methionine and 35S-cysteine​ (http://cbr.med.harvard.edu/investigators/springer/lab/protocols/jun_35Slabeling_IP.html)

Paola, your information posted contains a failure in the link. Could you put the right one? I would like to see it. Thanks.

try this

http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=2338

There are many protocols on web. Look for them by google

Anyway the Ebioscience trueblot, suggested from Olivier, is a good product

http://www.ebioscience.com/knowledge-center/product-line/trueblot-immunoblotting-technology.htm

Dear Amit,

I used successfully –as a secondary in the wb step- Protein A or Protein G HRP-conjugated (better for rabbit and mouse primary Abs, respectively) (Protein A GE brand, 1:5000, in my case, e.g.). These proteins should recognize the only not-reduced form of the Ab (so only the primary of the wb step, not the ip’s Ab run on the gel), but you need to test your specific Ab.

About the cross-linking (covalent binding), it is possible to covalently bind your Ab (that used for the the first step, the immunoprecipitation),

- or to Protein A/G agarose/sepharose beads, using an homobifunctional NHS-ester like the DSS (Disuccinimidyl suberate, 1.5-2mM final concentration) or DMP

- or to activated beads like Aminolink (from Thermo). In this case, the available amount of Ab should be higher, due to the random (not ‘directional’ as PA/PG) binding of the Ab (through the amines: K, R, …) (so a % of Ab is no more working after xl).

An additional care -for cross-linked resins- is to use –in the loading step- a laemli buffer without reductant (DTT or b-mercaptoethanol). Once you separated the supernatant from the beads, you can add the reductant to it.

Hope this helps, and keep us updated about your results.

Another way you can do is run it on non reducing condition on that way your Ab band will be shifted around 75 and you can cut the membrane and blott it up...

I would recommend using

Anti-IgG, Light Chain Specific

for Protein Detection on Western Blots after Immunoprecipitation

http://www.jacksonimmuno.com/catalog/CatPages/rblc.asp

works very well...only thing, sometime some protA leaks from protA matrix so , i may advice to prelute the beads with 0,2M glycine pHh 2 before doing the IP

Dear all,

since there are a lot of experts already in this conversation, I would like to ask what you would suggest to get rid of antibodies after a Co-IP if you are planning to do a Coomassie stain for MS instead of performing a western blot?

That's a tough one... If you know the epitope targeted by your IP antibody you could use an epitope peptide to try a specific elution. In mild conditions the Ab should stay on the beads, and the peptide would not be a problem for your downstream analysis given that it would go through the gel.

Dear Clemens, you can either do it the way Olivier suggested: like for example FLAG-tagged protein, FLAG IP and then FLAG peptide to elute the bound proteins from the beads. Alternatively, you can also just run your sample and cut your individual lane of your SDS PAGE gel into horizontal slices and perform MS from each slice. The slice containing the IgG bands will also give you other peptides whereas your IgG is not influencing all the other slices. Please take a look at our paper (Ramsey et al, Cancer Res 2011; Physical Association of HDAC1 and HDAC2 with p63 Mediates Transcriptional Repression and Tumor Maintenance in Squamous Cell Carcinoma) in which I did normal boiling of the beads in SDS-loading buffer after sequential FLAG and HA IP and performed a silver staining of the gradient gel (4-20%), cut out slices and send each for MS. The heavy chain of the IgG is labeled with an X. I hope this helps! Please ask if you have any other questions.

Good luck, Nicole

to Ip endogenous protein and if you have a lots of ab, the best is to crosslink the ab to prot A bead (the magnetic one, dynabeads are nice low background) with DSS or DMP (see pierce catalog). in this case, before performing your IP preluted the bead with glycine ph2,2...check the binding capacity of this affinity colum (ie how much of the prot you can pulld own with a fix vol of bead, ie compare the prot in the IP SN!), if your colum does not leak some ab...it is great and you can proceed to IP, afetr washing, it is best to elute the bound prot with acidic ph, people tends to use glycine, but think about 0,1%TFA, th eain advantage is that it is volatile and therefore, you can concentrate your eluate by speevacum:-)

if you are using a tagged prot, GFP tag is niec, because you can perform the pulldown with gfp trap (see angus lamond papers, there are the experts), it is a camel nanobody (13kda ) that run at the bottom ofthe gel and therefore it will not mask your prot partner

hope this help

I too think you should crosslink your antibody to any gel matrix you are using. You have very nice suggestions above. Another alternative is to use HRP-protein A instead of a secondary antibody. Protein A doesn’t bind single chain-denatured antibodies. But I would go for crosslinking.

I'm glad I found this post. I was panicking because I was IP'ing my overexpressed protein with mouse HA-beads, and one of the proteins I want to detect in my western is sitting at 50 kDA. However my antibody for this is anti-rabbit, so in theory the anti-rabbit secondary shouldn't cross react with the IgG chain. Not wanting the take the chance, I got my hands on some rabbit True blot and am awaiting the result now. Will update you all :)

have got good experience with TrueBlot.

Yes, I forgot to post with feedback after my last post, but I also got very nice results. Will be using it from now on!

Jackson Immuno Research has Light and Heavy chain specific secondary antibodies. So you would use the LC specific secondary to detect your protein around 55KD (you will not detect any heavy chain), and the HC specific for the lower molecular weight protein. (you will detect no light chain)I have had good luck with it.

I complete agree with Zachary. We have also found out the light chain specific antibody gives us very good results for detecting our IPed target protein in Western blot around 50 kD. It has been our routine to use this secondary antibody for other Western also. I highly recommend you give it a try. The one I used is HRP labled mouse anti-rabbit light chain specific secondary antibody. I use 1: 50,000 to 1:100,000. Definitely a must have in the lab if you need do a lot IP experiment.

Since we had a lot of problems with heavy chain lately (2 proteins were analyzed are around 50) and I looked at this post during our struggle, I would like to leave a short comment. Trueblot did not work well in our hands, neither mouse, nor rabbit (although their beads worked good for coIPs). So, we had to use antibodies from different species for coIP and detection. However, there is nice soft elution protocol that also helped (at least partially) to overcome the heavy chain problem:

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0018218

I have curious question on this.. When we use Elution buffer without DTT , there will be no heavy chain signal at all on western blott ??

Because when I am using LB without DTT i do get weak signal for heavy chain but I without DTT elution should not give any Heavy chain signal. I seem to not convince ..any inputs are welcome..

have you ever tested the TrueBlot sec Ab-HRP system? works very well as it will not recognize the heavyt or light chain. we use this on a regular basis as we are working with exosomes which have been isolated with Dynabeads and then processed for electrophoresis and western blotting. Many of the targets of interst have the same size as heavy or light chain which will make the interpretation difficult.

Use veriblot secondary antibody which doesn't detect denatured heavy and light chain 

I have ran into the similar problem, however, nor the non-reducing conditions or Jackson light chain-specific secondary Abs removed the problem. But I can confirm that the occurrence of those bands is not due to secondary Ab binding but due to unspecific binding of primary Ab, because when incubated only with the secondary Ab there was no signal on the blot. Maybe the True blot system will work better for this, but I have to test it.

I am interested in the secondary specific antibody for CoIP. I looked at the Jackson catalog but I did not find secondary anti HC antibodies, while I found anti LC ones. How do I search for anti HC?

Veriblot secondary antibody... 

easy blot is by far the best (1/100 ..1/200 dilution)

http://www.genetex.com/Web/News/NewsList.aspx?id=269

quite costly, but can resonnably reuse it upon freezing

I'm actually trying to solve this issue by blocking with unconjugated anti-rabbit-igG antibody, has anyone tried this?

@ Roudy Bo lawele Ekyalongo

One thing you need to do is to work under non reducing condition (  B-mercaptoethanol Free) this condition has been demonstrated by M. Yokota et al, at OSaka University,  your Ig molecule will migrate as tetramer having 150 kDa. so you can clearly detect your 55KDA or 25.

Do you have any references about this conclusion? Thanks.

The following are two papers, suggesting that heating can break or affect -S-S- WITHOUT reducing agents.

https://pdfs.semanticscholar.org/0fb7/771df062f6bc99db303aa5186f1f2b8a1082.pdf

http://pubs.rsc.org/en/content/articlelanding/2015/py/c5py00366k#!divAbstract

Please correct me if something is wrong. Thank you.

I am trying to troubleshoot a similar problem. I have tried using protein A-HRP. It has not worked for me. I have also tried eluting in non-reducing SDS cocktail and it has not worked. I have tried to better separate my proteins by running in a 4-12% gradient gel as well as by running a 14% gel for several hours. However, the signal from IgG is huge and it masks any nearby bands.

Next, I am planning to either try (1) Veriblot or (2)use light-chain specific Ab. Has anyone tried/compared options 1 and 2?

For a couple of years now, I have been using Trueblot ultra (Rockland) for mouse antibodies and ``conformation-specific HRP-labeled secondary`` from CST for rabbit antibodies, with good results.

Best, Manoj

I agree with Zachary T Kelleher suggestion. I have faced the similar problem with Heavy and Light chain bands in Co-immunoprecipitation masking my protein of interest at 27 KD and 54 KD respectively. However, after using LC specific secondary antibody for high molecular weight protein and HC specific secondary antibody for low molecular weight protein significantly increased the band intensity of my protein of interest.

Good luck..