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Tanzania, United Republic of
Antigua and Barbuda
Saint Kitts and Nevis
Saint Pierre and Miquelon
Trinidad & Tob
Korea, Rep of
Papua New Guinea
Bosnia and Herzegovina
Number of blots:
At least 20 (based on a 1:200 dilution in 5 ml milk).
Important protocol notes:
1. The VeriBlot for IP secondary antibody (HRP) detects the following IgG polyclonal and monoclonal antibodies:
|Human||IgG1, IgG2, IgG4|
|Mouse||IgG2a, IgG2b, IgG3|
2. The VeriBlot for IP secondary antibody (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.
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 ab131366 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||1/40 - 1/4000. The dilution will depend on the sensitivity of the HRP substrate. The dilution range recommended is 1:40 - 1:4000. Based on a 1:200 dilution (25 µL) in 5 ml milk researchers can perform 20 western blots. This product is recommended for the western blot detection of IP samples.|
ab128874 Immunoprecipitating Brd4 in human HEK293 whole cell lysate. 1000µg of cell lysate was incubated with primary antibody (1µg/mg in 50 mM Tris) and matrix (Protein G) for 16 hours at 4°C. For western blotting a HRP-conjugated Veriblot for IP secondary antibody (ab131366) (1/10000) was used to confirm successful immunoprecipation.
ab32371 immunoprecipitating Bak in human HCT116 p53-/- whole cell lysate. 100µg of cell lysate was incubated with primary antibody (1/100) and matrix (Protein A/G) for 4 hours at 4°C. For western blotting a HRP-conjugated Veriblot for IP secondary antibody (ab131366) (1/2000) was used to confirm successful immunoprecipation.
ab6148 Immunoprecipitating IRAK2 in human HEK293 whole cell lysate. 1000µg of cell lysate was incubated with primary antibody (1 µg/mg) and matrix (Protein G) for 16 hours at 4°C. For western blotting a HRP-conjugated Veriblot for IP secondary antibody (ab131366) (1/10000) was used to confirm successful immunoprecipation.
IP sample preparation: Histone H3 (mono methyl K9) was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to Histone H3 (mono methyl K9) and 50µl of protein G magnetic beads (+). No antibody was added to the control (-). The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation. Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC;
Western blot conditions: 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab9045.
Secondary antibody: IgG VeriBlot for IP secondary antibody (HRP) (ab131366) at 1/1000 dilution.
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