Can I use a purified mouse anti human cd63 that is for flow cytometry for western blot?
http://www.bdbiosciences.com/us/reagents/research/antibodies-buffers/immunology-reagents/anti-human-antibodies/cell-surface-antigens/pe-mouse-anti-human-cd63-h5c6/p/556020
I contacted the author of the paper and they responded "H5C6 is the name of the hybridoma clone that produces this anti CD63 antibody.
Whatever form it is sold under by BD, if the clone name is H5C6, it is the one we used"
I am still not getting any expression. Please help. Thank you
There may be two reasons:
In western blot the proteins are denatured. This allows some epitopes to be available for the antibody to bind but some antibodies bind better to the native form of the protein. It is very important to know which epitope is used for Ab production. If your antibody is designed only for flow cytometry analysis (it is possible that the antibody is recognizing a 3D epitope that is available in fixed cells), it may not be suitable for western blot applications.
Your BD antibody includes sodium azide which is one of the additives that is added by the vendors to stabilize the shelf life of antibodies. Sodium azide can interfere with HRP-conjugated antibodies which may effect your western blots, assuming that you are using an HRP detection system.
There may be two reasons:
In western blot the proteins are denatured. This allows some epitopes to be available for the antibody to bind but some antibodies bind better to the native form of the protein. It is very important to know which epitope is used for Ab production. If your antibody is designed only for flow cytometry analysis (it is possible that the antibody is recognizing a 3D epitope that is available in fixed cells), it may not be suitable for western blot applications.
Your BD antibody includes sodium azide which is one of the additives that is added by the vendors to stabilize the shelf life of antibodies. Sodium azide can interfere with HRP-conjugated antibodies which may effect your western blots, assuming that you are using an HRP detection system.
Sure, you an try to use it, maybe it works in both methods. (Try and error)
It depends. Several times antibodies for FC do not work in WB because they do not recognize denatured epitopes. Also please keep in mind that antibodies for FC can be more diluted than the antibodies for WB. Some vendors such as Cell Signaling they usually report if the antibody works for both FC and WB.
I am not sure how much your PE labelling disturbs in a Western blot application.
If a secondary antibody can still bind on the primary antibody.
A plain antibody without konjugate would likely work but with conjugate? No idea.
Depending on the detection system you commonly need HRP or AP conjugation or - if working with an infrared detection system - some fluorescence labelled antibody, wavelengh 700-800 nm.
how about the other way round? Can I use WB antibodies for flow cytometry? Deniz Cansen Kahraman
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