Far Western Analysis
http://www.piercenet.com/Proteomics/browse.cfm?fldID=BDAE338C-43BF-4FBA-B7D3-56C477BAEB9B
Protocol: http://onlinelibrary.wiley.com/doi/10.1002/0471142727.mb2006s55/pdf
Far Western Analysis is essentially a Western Blot, however instead of probing with a primary antibody, the blot (or even the gel directly) is probed with a "bait" protein conjugated to "tags". The protein to be detected on the blot is referred to as the "prey". Similar to a Western Blot, the probed membrane is then probed again, this time with an anti-body which recognizes the tag on the bait protein. This antibody may be conjugated to horse-radish peroxidase which, in the presence of hybrogen peroxide (H2O2) will catalyze the following reaction:
ROOR' + electron donor (2 e-) + 2H+ → ROH + R'OH
If the substrate (hybrogen peroxide) Horseradish peroxidase possessesh 4 lysine residues for conjugation to a labeled molecule. In the presence of the proper substrate, such as hydrogen peroxide, peroxidae activity occurs, producing a coloured, fluorimetric, or luminescent derivative of the labeled molecule when incubated with a proper substrate, allowing it to be detected and quantified.
http://www.piercenet.com/Proteomics/browse.cfm?fldID=BDAE338C-43BF-4FBA-B7D3-56C477BAEB9B
Protocol: http://onlinelibrary.wiley.com/doi/10.1002/0471142727.mb2006s55/pdf
Far Western Analysis is essentially a Western Blot, however instead of probing with a primary antibody, the blot (or even the gel directly) is probed with a "bait" protein conjugated to "tags". The protein to be detected on the blot is referred to as the "prey". Similar to a Western Blot, the probed membrane is then probed again, this time with an anti-body which recognizes the tag on the bait protein. This antibody may be conjugated to horse-radish peroxidase which, in the presence of hybrogen peroxide (H2O2) will catalyze the following reaction:
ROOR' + electron donor (2 e-) + 2H+ → ROH + R'OH
If the substrate (hybrogen peroxide) Horseradish peroxidase possessesh 4 lysine residues for conjugation to a labeled molecule. In the presence of the proper substrate, such as hydrogen peroxide, peroxidae activity occurs, producing a coloured, fluorimetric, or luminescent derivative of the labeled molecule when incubated with a proper substrate, allowing it to be detected and quantified.