Application
Immunoprecipitation
Sample
Mycobacterium tuberculosis Cell lysate - other (M. tuberculosis culture filtrate)
Total protein in input
1000 µg
Specification
M. tuberculosis culture filtrate
Immuno-precipitation step
Protein G
Other product details
Concentration
1 µg/mg lysate
Incubation time
16 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 0.05% TBS-T
Additional data
Abcam response
Thank you very much for your valuable feedback. We have not tested this antibody in IP, and it may not be suitable for this application.
I would recommend using protein A beads, as protein A has a higher affinity for rabbit immunoglobulins compared to protein G.
It appears that the secondary antibody has bound to the reduced rabbit IgG that was used in the IP, which appears as bands around 25 kDa (light chain) and 50 kDa (heavy chain). Although the secondary antibody would be anti-mouse, secondaries do often cross-react with IgG from similar species unless they are pre-adsorbed to ensure no cross-reactivity.
To reduce this background, you can use a secondary antibody that will only bind to non-reduced IgG following the Western blot with ab26246, such as ab131368.
I would recommend using protein A beads, as protein A has a higher affinity for rabbit immunoglobulins compared to protein G.
It appears that the secondary antibody has bound to the reduced rabbit IgG that was used in the IP, which appears as bands around 25 kDa (light chain) and 50 kDa (heavy chain). Although the secondary antibody would be anti-mouse, secondaries do often cross-react with IgG from similar species unless they are pre-adsorbed to ensure no cross-reactivity.
To reduce this background, you can use a secondary antibody that will only bind to non-reduced IgG following the Western blot with ab26246, such as ab131368.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
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投稿 Jul 11 2012