Hello Sajid,

Have you considered covalently cross linking your IgG antibody to your beads - that should keep them out of the eluate.

Here is a good link that might explain it for you:

(https://www.thermofisher.com/uk/en/home/life-science/protein-biology/protein-assays-analysis/immunoprecipitation/dynabeads-immunoprecipitation-crosslinking-protocol.html)

Good luck!

Best,

Peter

Hi, I don't have an easy answer for the light chain (25 kDa) band, but if you  use  Fab2 fragments (papain digestion of antibody to  remove FC potion) it should get red of the 55 kDa band. you can order Fab2 versions of many antibodies from suppliers.

Hi Sajid,

You can just use the secondary antibody against the light chain to avoid at least 55kD band if your protein of interest is around 55 kD. I have used the same for my work and it works fine. You can see the link.

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Hello Sajid,

Have you considered covalently cross linking your IgG antibody to your beads - that should keep them out of the eluate.

Here is a good link that might explain it for you:

(https://www.thermofisher.com/uk/en/home/life-science/protein-biology/protein-assays-analysis/immunoprecipitation/dynabeads-immunoprecipitation-crosslinking-protocol.html)

Good luck!

Best,

Peter

Raghib Bh,

As mentioned in my query, I incubated the membrane with Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 followed by anti-mouse IgG HRP-linked. Theoretically, I should not get any of the IgG bands when I use this antibody. 

I could not use light chain specific antibody, because I also need to probe for a protein which is around 25 kDa. 

Here, I am concerned whether this combination of antibodies is right if the primary antibodies that I am using for Immunoprecipitation are raised in mouse, whereas WB antibodies are raised in rabbit? What do you think?

Attached is the protocol that I followed with some experiment-specific modifications. Do you think its a good protocol to follow?

Hello Sajid,

you are technically right but this happens to me i tried the same as u mentioned but there was always a problem with heavy chain. So I decided to go for light chain antibodies. There is one more way i find at that time let me find I will let you know. 

your protocol is fine.

One more thing you have to include the bands of any of the chain (light or heavy) in your blot as a proof that you have used same amount of antibodies for immunoprecipitation in different treatment groups.

This paper will help your problem I think.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1350844/

Hi, Sajid,

I have faced the same problem in my study. I obtained clear signals by applying EasyBlot from GeneTex.

The easiest way is to use antibodies raised from different species for IP and WB. However, it is impossible in some cases. The commercial poly-clonal antibodies may be unworkable from different batches (I have met this situation).

The conformation-specific antibody from CST is not worked well in my hand. I also tried another popular secondary antibodies Trueblot; but it seems not so "true" to me.

I don't know whether it is acceptable or not in your lab to cut the membrane. If it is acceptable, you could cut your membrane then incubate the membrane containing proteins 50 kD with anti-light-chain-specific antibodies. I purchased these antibodies from JacksonImmunoResearch. 

Another solution that I have tried is incubation with HRP-conjugated protein G (The ref. listed below). However, the signals is much weaker than general secondary antibodies.

The product from Thermo seems to be HRP-conjugated protein A; but I am not sure. I just compared it to protein A by those antibodies that it can recognize.

Hope my experience to be benefit to your studies

Lal A, Haynes SR, Gorospe M. Clean Western blot signals from immunoprecipitated samples. Mol Cell Probes 2005;19:385-388.

Hi Shang-Hsin,

Thank you so much for sharing such useful information. Could you please provide here the catalogue numbers of anti-fc and anti-light chain specific secondary antibodies that you used?

Are these antibodies HRP-conjugated?

Thanks once again

Hi, Sajid,

The catalogue numbers: (The vendor is JacksonImmunoResearch; and all are HRP-conjugated)

*mouse anti-rabbit IgG, light-chain-sepcific; 211-032-171

*goat anti-rabbit IgG, Fc-fragment-specific; 111-035-046

*goat anti-mouse IgG, light-chain-specif; 115-035-174

*goat anti-mouse IgG (subclasses 1+2a+2b+3), Fc(gamma)-fragment-specifc; 115-035-164

After reconstitution and glycerol supplement (final conc. 50% glycerol), I use these antibodies at the titer of 1: 20,000~ 1: 30,000.

Hi Sajid Khan,

I presume you denature your sample before loading for western blot. If that is the case, I will suggest you use Veriblots Detection Reagent from Abcam as your secondary antibody (a 1:2500 in 5% milk in TBST dilution) should work fine for you. Veriblots Detection Reagent only detect native proteins and since you denature your sample during the elution step, the heavy and light chains whose disulfide bridge must have been broken should not be detected by Veriblots (which only detects native proteins). It is also not selective of the species in which your primary antibody was modeled in.

I hope this helps.

Good luck.

Hi Sajid Khan

As suggested by Michael, you can this reagent, we are using this reagent and it working fine.

In general IP-ed samples are reduced or denatured and then we will do SDS PAGE.

There is a reagent from abcam will detect only the native form of IgG but not the denatured IgG i.e heavy and light chains. Hence I can recommend this reagent

You can use this reagent from abcam to avoid the small and light chains of IgG.

VeriBlot for IP Detection Reagent (HRP) (ab131366)

You can follow the link below

https://www.abcam.com/veriblot-for-ip-detection-reagent-hrp-ab131366.html