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 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
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.
|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 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.
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.