Western blotting: Difference between revisions
Jump to navigation
Jump to search
Corrected the poorly done references and added some links. |
added link |
||
| Line 1: | Line 1: | ||
Western blotting is a core technique in the study of [[ | Western blotting is a core technique in the study of [[Molecular biology|molecular biology]] <ref>Serotec, A. (2015). Western Blot | What is Western Blotting?. [online] AbD Serotec. Available at: https://www.abdserotec.com/western-blotting.html [Accessed 2 Dec. 2015].</ref>. | ||
The technique is used to detect specific [[ | The technique is used to detect specific [[Proteins|proteins]] from a tissue extract; Western blots can also be used to evaluate the size of a protein of interest, and to measure the quantity of [[Protein expression|protein expression]] <ref>Towbin H, Staehelin T, Gordon J. (1979). "Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications". Proceedings of the National Academy of Sciences USA 76 (9): 4350–54. doi:10.1073/pnas.76.9.4350. PMC 411572. PMID 388439.</ref>. | ||
Gel electrophoresis is used initially to separate the proteins, next, they are stained with [[ | [[Gel_electrophoresis|Gel electrophoresis]] is used initially to separate the proteins, next, they are stained with [[Antibodies|antibodies]] specific to the target protein. | ||
From here, the first phase is known as the transfer step, what happens here is the movement of the proteins from solution to a biosynthetic membrane structure to form the 'blot' <ref>Bio-rad.com, (2015). Western Blotting | Applications &amp;amp; Technologies | Bio-Rad. [online] Available at: http://www.bio-rad.com/en-uk/applications-technologies/western-blotting [Accessed 2 Dec. 2015].</ref>. | From here, the first phase is known as the transfer step, what happens here is the movement of the proteins from solution to a biosynthetic membrane structure to form the 'blot' <ref>Bio-rad.com, (2015). Western Blotting | Applications &amp;amp;amp; Technologies | Bio-Rad. [online] Available at: http://www.bio-rad.com/en-uk/applications-technologies/western-blotting [Accessed 2 Dec. 2015].</ref>. | ||
The second stage uses a treatment which is known as blocking, this stage inhibits any non specific reactions from taking place. following this, the membrane is incubated with an antibody known as the [[ | The second stage uses a treatment which is known as blocking, this stage inhibits any non specific reactions from taking place. following this, the membrane is incubated with an antibody known as the [[Primary antibody|primary antibody]] which binds to the protein of interest. After this, any unbound primary antibody is washed away, and the membrane is incubated once again, however this time with a [[Secondary antibody|secondary antibody]] that specifically recognises and binds to the primary antibody. The secondary antibody is linked to a reporter enzyme that produces colour allowing it to be easily detected and imaged <ref>Nature.com, (2015). Western blot | Learn Science at Scitable. [online] Available at: http://www.nature.com/scitable/definition/western-blot-288 [Accessed 2 Dec. 2015].</ref>. | ||
=== References === | === References === | ||
<references /><br> | <references /><br> | ||
Revision as of 15:07, 28 November 2017
Western blotting is a core technique in the study of molecular biology [1].
The technique is used to detect specific proteins from a tissue extract; Western blots can also be used to evaluate the size of a protein of interest, and to measure the quantity of protein expression [2].
Gel electrophoresis is used initially to separate the proteins, next, they are stained with antibodies specific to the target protein.
From here, the first phase is known as the transfer step, what happens here is the movement of the proteins from solution to a biosynthetic membrane structure to form the 'blot' [3].
The second stage uses a treatment which is known as blocking, this stage inhibits any non specific reactions from taking place. following this, the membrane is incubated with an antibody known as the primary antibody which binds to the protein of interest. After this, any unbound primary antibody is washed away, and the membrane is incubated once again, however this time with a secondary antibody that specifically recognises and binds to the primary antibody. The secondary antibody is linked to a reporter enzyme that produces colour allowing it to be easily detected and imaged [4].
References
- ↑ Serotec, A. (2015). Western Blot | What is Western Blotting?. [online] AbD Serotec. Available at: https://www.abdserotec.com/western-blotting.html [Accessed 2 Dec. 2015].
- ↑ Towbin H, Staehelin T, Gordon J. (1979). "Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications". Proceedings of the National Academy of Sciences USA 76 (9): 4350–54. doi:10.1073/pnas.76.9.4350. PMC 411572. PMID 388439.
- ↑ Bio-rad.com, (2015). Western Blotting | Applications &amp;amp; Technologies | Bio-Rad. [online] Available at: http://www.bio-rad.com/en-uk/applications-technologies/western-blotting [Accessed 2 Dec. 2015].
- ↑ Nature.com, (2015). Western blot | Learn Science at Scitable. [online] Available at: http://www.nature.com/scitable/definition/western-blot-288 [Accessed 2 Dec. 2015].