Western blot
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- Опубликовано: 16 сен 2024
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The western blot (sometimes called the protein immunoblot) is a widely accepted analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide. The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are stained with antibodies specific to the target protein.[1][2]
There are now many reagent companies that specialize in providing antibodies (both monoclonal and polyclonal antibodies) against tens of thousands of different proteins.[3] Commercial antibodies can be expensive, although the unbound antibody can be reused between experiments. This method is used in the fields of molecular biology, biochemistry, immunogenetics and other molecular biology disciplines.
Other related techniques include using antibodies to detect proteins in tissues and cells by immunostaining and enzyme-linked immunosorbent assay (ELISA).
The method originated in the laboratory of Harry Towbin at the Friedrich Miescher Institute.[1] The name western blot was given to the technique by W. Neal Burnette[4] and is a play on the name Southern blot, a technique for DNA detection developed earlier by Edwin Southern. Detection of RNA is termed northern blot and was developed by George Stark at Stanford. Source of the article published in description is Wikipedia. I am sharing their material. Copyright by original content developers of Wikipedia.
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~3-5 % BSA (Bovine serum albumine) in PBS ( Phosphate Buffer Solution) or Non-fat dry milk in buffers like PBS or Tris buffered solution are the commonly used blocking agents.
~Blocking agents are used to prevent the non-specific binding of antibody to the to the blotting membrane.
~Detergents like Tween20 is also introduced that helps in reduction of background stain.
thanks for putting this together, it was a big help to me!
you have a good speaking voice
Great video describing WB in simple and clear terms.
This was an awesome explanation!
fantastic lecture i understood with less effort thank youuuuuuu
nice explanation...but u should clarify some more abbreviations like pvdf, and why luminol is used...significance of it etc. no intention to criticize at all...just giving u a suggestion.. otherwise it z very easy to understand
Thank you Sir, this video was helpful
You're welcome
Its a very informative video. Thank you.
How blocking is carried out?
what are the reagents used for it?
pls give such detailed explanation for those steps.
thank u sumanji......
Thank you for your lecture, great!
very helpful and simple
thanxxx.....
very informative.....
Loved it! #readyforcellbiolabfinal
thank you..the video ws informative
good presentation
Since both the antibody which we are using taken from somewhere, then why we only adding enzyme with secondary antibody, there is a primary abtibody present why we are not incorporating the enzyme with primary abtibody, this will save money
Thank You!
awesome . thank u so much sir
thank you.
this is helpful
Antibodies for antibodies. Mind blown. Why not just modify the rabbit antibody so it has the detectable marker?
Edit: Nvm, you answer my question in the video
thank u sir
why do we do WB when ELISA can do the same thing, more rapid and simple?
the last step is almost same as ELISA procedure... don't understand why do we need to denature proteins and fix them in nitrocellulose membrane...
six years later, did you find answers?
Helpful, but there's no Step 4 haha
Almost all WB videos have a Indian explaining lol