Overview

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C.
  • Storage buffer

    pH: 7.2
    Constituents: 0.16% Sodium phosphate, 0.88% Sodium chloride, 50% Glycerol, 0.1% BSA
  • Concentration information loading...
  • Clonality

    Monoclonal
  • Isotype

    IgG
  • General notes

    Number of blots:
    At least 20 blots, based on a 1 µl/ml dilution and 5 ml diluted antibody per blot.

    Important protocol notes:

    1. The anti-mouse IgG for IP secondary antibody (HRP) detects mouse IgG antibodies (subtypes: IgG1, IgG2a, IgG2b, IgG3).

    2. The anti-mouse IgG for IP secondary antibody (HRP) preferentially detects the non-reduced form of mouse IgG (IgG1, IgG2a, IgG2b, IgG3) over the reduced, SDS-denatured forms.

    3. IP sample should be completely reduced/denatured before loaded onto a western blot.

    4. Milk should be used as the blocking protein for the immunoblot.

    Western blot and IP resources:
    a) Western blot a beginner's guide
    b) IP protocol
    c) IP troubleshooting tips

  • Research areas

Applications

Our Abpromise guarantee covers the use of ab131368 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/1000 - 1/10000.

The optimal dilution will depend on the sensitivity of the HRP substrate.

Images

  • IP sample preparation: Caspase 7 was immunoprecipitated from 0.5 ml of 1x107 Jurkat (Human T cell leukemia cell line from peripheral blood) cells/ml with 5 µg mouse anti-human Caspase 7.

    WB conditions: Precipitate from 1x106 cells was subjected to electrophoresis, transferred to an PVDF membrane, and immunoblotted with an anti-Caspase 7 antibody.

    Detection:
    Lane 1: Detection with anti-mouse IgG for IP secondary antibody (HRP) (ab131368)
    Lane 2: Detection with an HRP-conjugated anti-mouse IgG H&L secondary antibody
    Lane 3: Lane 1 was re-immunoblotted using an HRP-conjugated anti-mouse IgG H&L secondary antibody. The heavy and light-chains can now be seen, confirming that although the immunoprecipitating heavy and light-chains are present, ab131368 detects only native antibody and not denatured heavy and light-chains.

    Please note the detection of the heavy and light-chains of the immunoprecipitating antibody in Lane 2 but not in Lane 1.

Protocols

References

This product has been referenced in:

  • Scrivo A  et al. Gigaxonin E3 ligase governs ATG16L1 turnover to control autophagosome production. Nat Commun 10:780 (2019). Read more (PubMed: 30770803) »
  • Liu J  et al. Induction of entosis in prostate cancer cells by nintedanib and its therapeutic implications. Oncol Lett 17:3151-3162 (2019). Read more (PubMed: 30867745) »
See all 60 Publications for this product

Customer reviews and Q&As

1-10 of 11 Abreviews or Q&A

Abreviews
Application
Western blot
Works well
The IP was done with a mouse antibody and afterwards WB was also done with a mouse antibody

Abcam user community

Verified customer

Submitted Oct 03 2016

Abreviews
Application
Western blot
IRAK2 was immunoprecipiated from 1 mg HEK293 WCE, run on an 8% gel and western blotted with the same antibody. The veriblot secondary antibody was used at a concentration of 1/10000 for 1 hr at room temp before detection by ECL.

Abcam user community

Verified customer

Submitted Nov 06 2013

Answer

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

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Answer

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.

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Answer

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.

Read More

Answer

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.

Use our products? Submit an Abreview. Earn rewards!
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Read More

Answer

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.

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Answer

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.

Read More

Answer

Thank you for contacting us. I am sorry that this antibody is detecting multiple bands in WB on rat samples.
Is the secondary antibody you are using pre-adsorbed against rat? It is possible that the anti-mouse secondary may be detecting the heavy and light chains of endogenous IgGs. The heavy and light chains tend to run around 50 and 25 kDa respectively, but variations in buffer system and sample preparation could raise the observed molecular weights and this may account for the bands around 60 and 27 kDa. I would recommend using a preadsorbed secondary antibody, or a secondary antibody that is designed to only detect native IgGs, such as our new VeriBlot range (ab131368 for example).
I would also recommend loading no more than 50 ug of protein to ensure the maximum specificity possible, especially when using a very sensitive ECL reagent.
I hope this helps to improve your results, if not, I will be happy to offer you a free of charge replacement, credit, or refund as long as you have purchased the antibody in the past six months. Please let me know your original order number and how you would like to proceed and I will be happy to help you further.

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1-10 of 11 Abreviews or Q&A

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