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Western blotting


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#1 lvhongyu

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Posted 23 December 2009 - 02:51 AM

Antibodies to most proteins can be created by injecting small amounts of the protein into an animal such as a mouse, rabbit, sheep, or donkey (polyclonal antibodies)or produced in cell culture (monoclonal antibodies). These antibodies can be used for a variety of analytical and preparative techniques.

In western blotting, proteins are first separated by size, in a thin gel sandwiched between two glass plates in a technique known as SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). The proteins in the gel are then transferred to a PVDF, nitrocellulose, nylon or other support membrane. This membrane can then be probed with solutions of antibodies. Antibodies that specifically bind to the protein of interest can then be visualized by a variety of techniques, including colored products, chemiluminescence, or autoradiography. Often, the antibodies are labeled with an enzymes. When a chemiluminescent substrate is exposed to the enzyme it allows detection. Using western blotting techniques allows not only detection but also quantitative analysis.

Analogous methods to western blotting can be used to directly stain specific proteins in live cells or tissue sections. However, these immunostaining methods, such as FISH, are used more often in cell biology research.

#2 Prep!

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Posted 23 December 2009 - 03:34 AM

Antibodies to most proteins can be created by injecting small amounts of the protein into an animal such as a mouse, rabbit, sheep, or donkey (polyclonal antibodies)or produced in cell culture (monoclonal antibodies). These antibodies can be used for a variety of analytical and preparative techniques.

In western blotting, proteins are first separated by size, in a thin gel sandwiched between two glass plates in a technique known as SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). The proteins in the gel are then transferred to a PVDF, nitrocellulose, nylon or other support membrane. This membrane can then be probed with solutions of antibodies. Antibodies that specifically bind to the protein of interest can then be visualized by a variety of techniques, including colored products, chemiluminescence, or autoradiography. Often, the antibodies are labeled with an enzymes. When a chemiluminescent substrate is exposed to the enzyme it allows detection. Using western blotting techniques allows not only detection but also quantitative analysis.

Analogous methods to western blotting can be used to directly stain specific proteins in live cells or tissue sections. However, these immunostaining methods, such as FISH, are used more often in cell biology research.



thanx for the information Iv..... anyways wud like to add one more thing here that the WB is not only co-related to SDS-PAGE but can be done after IEF (Iso-electric focussing also) which will be based on charge. WB has many applications. It is a separate technique and not always in-sync with SDS-PAGE.
You can share more but it can be more informative if its a discussion or a querry tat u ve faced or anything tat u have observed abt the technique tat u might want to share with us which might help us in ours...
Thanx
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#3 mdfenko

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Posted 23 December 2009 - 08:54 AM

hey, pi, this is just ivhongyu's way of introducing a hidden link (notice the underlined "gel") into the forum. the link itself may be useful to us but i really dislike the attempt to fool us into accessing the website.

i wish that those who have useful websites would just post them in the proper forum and not try to "put one over on us".

Edited by mdfenko, 23 December 2009 - 08:54 AM.

talent does what it can
genius does what it must
i do what i get paid to do

#4 Prep!

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Posted 23 December 2009 - 07:04 PM

:P
Support bacteria - They are the only culture some people have!!!
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#5 HomeBrew

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Posted 23 December 2009 - 08:35 PM

What link? :D




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