Western Blot
This technique permits identification of proteins by their molecular weight and their immunoreactivity or the control of the immunoreactivity of serum on the denaturated protein of interest.
•1st step: Separation of customer’s purified protein fraction by electrophoresis
in 10%* PAGE under denaturing conditions then transfer to nitrocellulose
membrane.
•2nd step: Saturation then incubation of membrane with the diluted serum.
•3rd step: Washing of nitrocelllulose membrane and incubation with an
antiserum labeled with HRP.
•4th step: Washing then revelation of HRP activity by chemiluminescence with Covalab’s detection kit: Covalight® (Ref.: opr0008, opr0009).
During the revelation any photons emitted are detected on a photographic
film. Proteins appear as dark bands on the film.
* The percentage of polyacrylamide gel slice can be modified according to
the molecular weight of protein.
Type of sample:
Purified protein: 15 – 20 µg
Cellular lysate containing the protein of interest:
500 µg
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