Dear Guang Lu,

I had once the same problem. My solution was to use a special secondary antibody which is not detecting the heavy chain. They are available from different companies. 

I guess you're using ECL for detecting your bands? 

In case you're using an IR system, there are also other solutions, but this was what I did for using ECL. It was working quite fine!

I hope this helps you! 

Dear Guang Lu,

I had once the same problem. My solution was to use a special secondary antibody which is not detecting the heavy chain. They are available from different companies. 

I guess you're using ECL for detecting your bands? 

In case you're using an IR system, there are also other solutions, but this was what I did for using ECL. It was working quite fine!

I hope this helps you! 

Hi Guang Lu, I support what Arianne suggests, you will need to use secondary antibodies that only recognize native primary antibodies (what you use for IP is denatured in SDS loading buffer for the PAGE). They are called TrueBlot or VeriBlot or similar names (I attach a few links...).

Other option is to use a primary against your protein raised in a different species, since secondary species specificity is pretty good. For example I was IPing one protein with a mouse monoclonal and then identifying it on WB with a chicken polyclonal... Of course there is not always this possibility...

Good luck!

http://www.abcam.com/secondary-antibodies/secondary-antibodies-optimized-for-co-immunoprecipitation-western-blotting

http://www.rockland-inc.com/TrueBlot-IP-Western-Blotting-Main.aspx

http://www.tebu-bio.com/techinfo/878/TrueBlot%C2%AE_Clear_Detection_of_Blotted_Target_Bands.html

Hi there,

Use antibodies raised in different hosts for IP and WB. An alternative would be to use agarose beads covalently conjugated with Antibody for CoIP.

Hi,  Ariane Eberhardt and  Pietro Pilo Boyl, thanks very much for your answers. I think your suggestions should be very useful. I previous used a Light chain second anti-body, but I still can detect both chains of the IgG. I don't know whether this is the similar antibody you mentioned. Which brand you ever used when you detect the immunoprecipitates?

Hi, Dominique Liger. Thanks for your answer. I did perform a Flag IP with good result. But this time I need to detect the endogenous protein level and thus the IgG problem can not be avoided. 

Dear Guang Lu,

just check out the ImmunoCruz IP/WB Optima System from Santa Cruz. I was using this quite successfully. 

Dear Guang Lu,

The problem may be avoided if you have a specific antibody raised against your untagged protein of interest. It's about conjugating it covalently to agarose beads. There are different approaches to generate such conjugates, read the following for an overview:

https://www.thermofisher.com/fr/fr/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/covalent-immobilization-affinity-ligands.html

Hi Guang Lu, theoretically you were correct, since your protein of interest runs with 55 kDa apparent MW, using Abs against the light chain only would be sufficient. Why this didn't work in your hands I cannot say, possibly the Company did not sell as specific Abs as they advertise... it is not unusual.

In any case there are Abs against the light chain that recognize both denatured and native light chains and more specific secondaries that only recognize native light chains, which therefore remove also the signal at 25 kDa (in your case probably not so important). These last ones are the ones I was indicating in the previous post. We specifically use the Rockland TrueBlot secondaries, but in principle I would trust also Abcam VeriBlot, and of course, although I don't particularly favour Santa Cruz antibodies, the Optima System will be fine since it was successfully used by Ariane.

Just a comment on Dominique's suggestion. The binding of the Ab to activated sepharose beads is of course a possibility, but with an eye to the type of activated beads. For example the use of the most common CNBr-activated sepharose would not be a good idea because the isourea derivative that forms upon binding of primary amines from the Ab hydrolyzes when boiled in SDS buffer, releasing the Ab. But of course NHS or other activating groups that form amide bonds or aminic bonds will be suitable.

Good luck.

Good points raised by Pietro.  An alternative you may consider if you do not want to invest in Abs specific for native IgG  is to denature your samples without reducing them before loading.  The IgG may still be released from the beads (if covalently attached by isourea linkages or not covalently crosslinked) but will migrate mostly as high molecular weight bands ( about 150 kDa); note that some monomeric heavy chain may still be seen though, depending on your Ab, so make sure you have some Ab alone control.  This approach will also reduce background with native 'specific' antibodies, which may still react weakly with denatured IgG.  Of course if you must reduce your samples, you better stick with Trueblot alternatives (but I would still include an Ab alone control, just in case).

Good luck!

What I am generally doing when I go for Co-IPs with proteins coming in the range of heavy or light chains or generally when I want to get some MS analysis after Co-IP is that I go for a magnetic antibody-crosslinking first which removes approx. 95-98% of the heavy and light chains. https://www.thermofisher.com/order/catalog/product/88805 The method is easy, takes obviously a few hours but it is highly efficient and solved a lot of problems for me.

Dear all, thanks very much! Most of your suggestions are very useful. I think I will first try Rockland TrueBlot secondaries. 

Guang Lu

Hi, Guang.

I have exactly the same problem now. Do you mind let me know which methods did you choose before? And did it work well? I am looking forward to your response.

Thank you very much