VeriBlot for IP Detection Reagents are immunoblotting reagents that enable the trouble-free detection of immunoblotted target protein bands, without interference from denatured IgG. This allows to detect the (co-)immunoprecipitated protein without masking by the IgG heavy (50 kDa) and light chains (25 kDa). In general, this interference tends to originate from secondary antibodies which recognize primary antibodies released with the antigen during the immunoprecipitation procedure or endogenous IgGs from the lysate itself. VeriBlot for IP detection reagents only recognize native (non-reduced) antibodies and therefore the detection of heavy and light chains is highly minimized, if the immunoprecipitate is fully reduced.
Number of blots:
At least 20 (based on a 1:200 dilution in 5 ml milk).
Important protocol notes (This information is available in Chinese here)
1. The VeriBlot for IP Detection Reagent (HRP) detects the following IgG polyclonal and monoclonal antibodies:
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Species
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Monoclonal Isotype(s)
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Bovine
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IgG2
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Goat
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IgG2
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Human
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IgG1, IgG2, IgG4
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Mouse
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IgG2a, IgG2b, IgG3
Note: If using mouse IgG1, perform a dot blot to determine compatibility. VeriBlot for IP Detection Reagent (HRP) might not detect mouse IgG1.
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Rat
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IgG2C
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Rabbit
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Total IgG
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Sheep
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IgG2
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2. The VeriBlot for IP Detection Reagent (HRP) preferentially detects the non-reduced form over the reduced, SDS-denatured forms.
3. IP sample should be completely reduced/denatured before loaded onto a western blot. Boil samples for 5-10 minutes in SDS sample buffer with a increase in SDS amount if required.
4. Milk should be used as the blocking protein for the immunoblot.
Note: If denatured and blotted IgG are not clearly detected, the following steps may be used to increase the amount of denatured IgG in the sample:
- Increase the concentration of reducing agent
- Boil sample to aid in reduction of IgG disulfide bonds
- Use dentaturing electrophoresis conditions
A full troubleshooting guide is available here.
