Number of blots: At least 20 blots, based on a 1 µl/ml dilutionand 5 ml diluted antibody per blot. Important protocol notes: 1. Theanti-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
Our Abpromise guarantee covers the use ofab131368in 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. |
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 5µg 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 antibodyLane 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.
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ab131368 has been referenced in 82 publications.
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