Anti-mouse IgG VeriBlot for IP secondary antibody (HRP) (ab131368)

Overview

Properties

  • FormLiquid
  • Storage instructionsStore at +4°C.
  • Storage bufferpH: 7.2
    Constituents: 0.16% Sodium phosphate, 0.88% Sodium chloride, 50% Glycerol, 0.1% BSA
  • Concentration information loading...
  • ClonalityMonoclonal
  • IsotypeIgG
  • General notes

    Number of blots:
    At least 20 blots (based on a 1 µl/ml dilution in 5 ml milk).

    Important protocol notes:

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

    2. The anti-mouse VeriBlot 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 Use at an assay dependent concentration. The dilution will depend on the sensitivity of the HRP substrate. The recommended dilution is 1 µl in 1 ml milk. Based on the recommended dilution in 5 ml milk researchers can perform 20 western blots. This product is recommended for the western blot detection of IP samples.

Anti-mouse IgG VeriBlot for IP secondary antibody (HRP) images

  • IP sample preparation: Caspase 7 was immunoprecipitated from 0.5 ml of 1x107 Jurkat cells/ml with 5 ug 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 VeriBlot 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.

  • Developed using ECL technique under reducing conditions; exposure time 3 mins; blocking and antibody incubation steps done in 5% milk/TBST
    Lanes:
    1: Marker
    2: Human heart homogenate Tissue Lysate – 20 µg
    3: HeLa Cell Lysate – 20 µg
    4: Mouse heart homogenate Tissue Lysate - 20 µg
    5: NIH3T3 Cell Lysate – 20 µg
    6: Rat heart homogenate Tissue Lysate – 20 µg
    7: H9C2 cell lysate – 20 µg

    All Lanes:
    Anti-Sodium ATPase antibody – Plasma Membrane Marker
    Anti-GAPDH antibody – Cytosolic Marker
    Anti-Histone H3 (di methyl k9) antibody – Nuclear Marker

    Secondary: Anti-Mouse IgG VeriBlot for IP secondary antibody (ab131368) at 1/1000 dilution

    Predicted Sodium Potassium ATPase band size: 112 kDa
    Observed Sodium Potassium ATPase band size: 100 kDa
    Predicted GAPDH band size: 37 kDa
    Observed GAPDH band size: 37 kDa
    Predicted Histone H3 (di methyl k9) band size: 17 kDa
    Observed Histone H3 (di methyl k9 band size: 17 kDa

Protocols

References for Anti-mouse IgG VeriBlot for IP secondary antibody (HRP) (ab131368)

This product has been referenced in:
  • Januschke J  et al. Centrobin controls mother-daughter centriole asymmetry in Drosophila neuroblasts. Nat Cell Biol 15:241-8 (2013). Read more (PubMed: 23354166) »

See 1 Publication for this product

Product Wall

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.
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Verified customer

Submitted Nov 06 2013

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 mou...

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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 w...

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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 coul...

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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 th...

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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 ...

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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 specifi...

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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 ...

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Thank you for your feedback! We are always happy to help, and if you have any further questions please let me know.

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"