Prab: CSV import

2024-04-22T04:57:33Z

CSV import

New page

[[File:Western_blot_workflow.jpg|Western blot workflow|thumb]] [[File:Western_Blot_results_for_HIV_test.jpg|Western Blot results for HIV test|thumb|left]] [[File:Journal.pbio.3001783.g001.tif|Journal.pbio.3001783.g001|thumb|left]] [[File:SDS-PAGE_Electrophoresis.png|SDS-PAGE Electrophoresis|thumb]] [[File:Western_blot_transfer.png|Western blot transfer|thumb]] [[File:Western_Blot_binding.png|Western Blot binding|thumb]] '''Western blot''' (also known as protein immunoblot) is a widely used [[analytical technique]] in [[molecular biology]] and [[immunogenetics]] to detect specific [[proteins]] in a sample of tissue homogenate or extract. The method uses [[gel electrophoresis]] to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/non-denaturing conditions). The proteins are then transferred to a membrane (typically [[PVDF]] or [[nitrocellulose]]), where they are stained with antibodies specific to the target protein.

==Procedure==
The Western blot procedure involves several steps:
# Sample preparation: The protein samples are prepared by mixing with a [[SDS-PAGE]] loading buffer and possibly heating.
# [[Gel electrophoresis]]: The proteins are separated based on their size through SDS-PAGE.
# Transfer: The proteins are transferred from the gel onto a membrane, making them more accessible for analysis.
# Blocking: The membrane is incubated with a solution of protein (such as non-fat dry milk) to block nonspecific binding sites.
# Antibody incubation: The membrane is incubated with a primary antibody specific to the target protein. Then, after washing, it is incubated with a secondary antibody conjugated to a reporter enzyme or dye.
# Detection: The enzyme or dye is activated, producing a signal that can be detected by various methods, indicating the presence and quantity of the target protein.

==Applications==
Western blotting is used in both scientific research and medical diagnostics. In research, it helps in identifying specific proteins in a complex mixture and studying protein expression under various conditions. In diagnostics, it is employed for the detection of [[antibodies]] in diseases like [[HIV]], [[Lyme disease]], and [[Hepatitis B]].

==Advantages and Limitations==
The main advantage of Western blotting is its specificity; it can detect a single protein in a mixture of thousands of proteins. However, it is a time-consuming process and requires a significant amount of technical skill to perform accurately. Additionally, quantification can be challenging and less precise compared to other methods like [[ELISA]].

==See Also==
* [[ELISA]]
* [[Immunohistochemistry]]
* [[Northern blot]]
* [[Southern blot]]

[[Category:Biochemistry methods]]
[[Category:Molecular biology]]
[[Category:Immunologic tests]]
{{stub}}

Western blot - Revision history

Prab: CSV import