Anti-mouse IgG for IP (HRP) (ab131368)

ab131368 has only been tested in a gel with SDS, but it should detect non-reduced mouse IgG run with or without SDS as it binds to the non-reduced native mouse IgG primary antibody that is used to probe the SDS-PAGE blot

We do not know where the epitope this rat monoclonal antibody recognizes is located on mouse IgG, whether light chain or heavy chain, or if it contains amino acids from the light and also the heavy chain. We do know that the antibody will recognize mouse IgG containing either kappa or lambda light chain, and that the epitope is present only on non-denatured IgG. If the epitope contains light chain amino acids, it is assumed they are identical in kappa and lambda.

Thank you very much for the time taken giving me furthrt infotmation.

I can confirm that the Goat F(ab')2 polyclonal Secondary Antibody to Mouse IgG - (Fab')2 (HRP), pre-adsorbed could overcome the cross-reactivity with the human Fc. However we regrattably cannot give a guarantee for it.

If the immunoblot is performed under denatured and reduced conditions I would recommend to have a look at our anti native antibodies (VeriBlot). This secondary antibodies will not react with denatured proteins. For the use of this antibodies it is improtant to have fully denatured samples. The blocking should be performed with milk (Please read the data sheet carefully). If you https://www.abcam.com/mouse-igg-veriblot-for-ip-secondary-antibody-hrp-ab131368.htmlyou will find the Anti-mouse IgG VeriBlot for IP secondary antibody (HRP) - ab131368 that might be suitable for the experiment.

I hope this information is helpful. If you have further questions please do not hesitate to contact us again.

Thank you for contacting us.

When using ab21899 Anti-beta 2 Microglobulin antibody [B2M-01] (Biotin) in IP we can recommend a working range of 1-10 ug/ml. The exact concentration for your IP experiments should be within this range but could be outside of this and will need to be determined experimentally.

Abcam also provides a number of products for use in IP such as our https://www.abcam.com/ab119211 ready to use protein gel stain, our https://www.abcam.com/index.html?pageconfig=resource&rid=15115#1 and our https://www.abcam.com/index.html?pageconfig=resource&rid=15115#2 . Our VeriBlot line only recognize native (non-reduced) antibodies and are recommended when the target of interest is <30kDa, whereas our Light Chain specific line do not recognize heavy-chains and are recommended when your protein of interest is >30kDa. All of our products are covered by our six month Abpromise guarantee and are fully supported by our scientific support staff. More information about these products can be found at the links below.

Optiblot Blue: https://www.abcam.com/Optiblot-Blue-ab119211.html?refer404=catalogue%20119211

IP specific secondary antibodies: https://www.abcam.com/index.html?pageconfig=resource&rid=15115


I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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There is a protocol note for ab131368 that states:
"4. Milk should be used as the blocking protein for the immunoblot."
I suggest using milk as the blocking buffer in efforts to reduce background on the blots.
Also, please ensure that the samples loaded onto the gel are fully reduced prior to loading.

Thank you for your enquiry and for including all your data. That was very helpful.
Regarding Figure 1:
a. In which cell lines do you expect Glutaminase to be expressed?,
b. background is quite high in both figures 1 and 3. What blocking buffer was used?
Regardomg Figure 3:
a. What are molecular weights of molecular weight marker bands?
b. What does "before" and 'After" refer to? in page 3 and 4?
c. Have you done a 'no primary' control blot?
Overall it looks like no antibody specificity because commassie stain looks just like WB images.

Thank you for contacting us. I am sorry that ab8984 is giving poor results in WB.

Have you validated your transfer at the higher molecular weights by Ponceau stain? If the transfer is incomplete, that could account for the lack of the specific band. You could try transferring longer or adding 0.1% SDS to the transfer buffer and lowering the methanol percentage to 10%.

If you are able to detect the specific band with these alterations, you may be able to reduce some of the background bands. The bands around 25 and 50 kDa may be due to the secondary antibody cross-reacting with endogenous IgGs (this can be checked by running a no primary antibody control). If you are getting background due to endogenous IgGs, you could switch to a secondary antibody that is specific for native IgGs, such as our VeriBlot secondary ab131368.

Could you please let me know what dilutions and incubation times you were using for your primary and secondary antibodies? What blocking agent was used and at what percentage?

I hope this helps, if not, I will be happy to offer you a free of charge replacement, credit, or refund. Please let me know your original order number and how you would like to proceed and I will be happy to help you help you further.