Electroblotting is a method in

molecular biology Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and phys ...

biochemistry Biochemistry or biological chemistry is the study of chemical processes within and relating to living organisms. A sub-discipline of both chemistry and biology, biochemistry may be divided into three fields: structural biology, enzymology ...

immunogenetics Immunogenetics or immungenetics is the branch of Medical Immunology and Medical Genetics that explores the relationship between the immune system and genetics. Autoimmune diseases, such as type 1 diabetes, are complex genetic traits which res ...

to transfer

proteins Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respondi ...

nucleic acids Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main cl ...

onto a membrane by using

PVDF Polyvinylidene fluoride or polyvinylidene difluoride (PVDF) is a highly non-reactive thermoplastic fluoropolymer produced by the polymerization of vinylidene difluoride. PVDF is a specialty plastic used in applications requiring the highest pur ...

nitrocellulose Nitrocellulose (also known as cellulose nitrate, flash paper, flash cotton, guncotton, pyroxylin and flash string, depending on form) is a highly flammable compound formed by nitrating cellulose through exposure to a mixture of nitric acid and ...

, after

gel electrophoresis Gel electrophoresis is a method for separation and analysis of biomacromolecules ( DNA, RNA, proteins, etc.) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size (IEF ...

. The protein or nucleic acid can then be further analyzed using probes such as specific antibodies,

ligand In coordination chemistry, a ligand is an ion or molecule ( functional group) that binds to a central metal atom to form a coordination complex. The bonding with the metal generally involves formal donation of one or more of the ligand's ele ...

s like

lectin Lectins are carbohydrate-binding proteins that are highly specific for sugar groups that are part of other molecules, so cause agglutination of particular cells or precipitation of glycoconjugates and polysaccharides. Lectins have a role in rec ...

s, or stains. This method can be used with all polyacrylamide and agarose gels. An alternative technique for transferring proteins from a gel is capillary blotting.

Development

This technique was patented in 1989 by William J. Littlehales under the title "Electroblotting technique for transferring specimens from a polyacrylamide electrophoresis or like gel onto a membrane.

Electroblotting procedure

This technique relies upon current and a transfer buffer solution to drive proteins or nucleic acids onto a membrane. Following electrophoresis, a standard tank or semi-dry blotting transfer system is set up. A stack is put together in the following order from cathode to anode: sponge , three sheets of filter paper soaked in transfer buffer , gel , PVDF or nitrocellulose membrane , three sheets of filter paper soaked in transfer buffer , sponge. It is a necessity that the membrane is located between the gel and the positively charged anode, as the current and sample will be moving in that direction. Once the stack is prepared, it is placed in the transfer system, and a current of suitable magnitude is applied for a suitable period of time according to the materials being used. Typically the electrophoresis gel is stained with

Coomassie brilliant blue Coomassie brilliant blue is the name of two similar triphenylmethane dyes that were developed for use in the textile industry but are now commonly used for staining proteins in analytical biochemistry. Coomassie brilliant blue G-250 differs fr ...

following the transfer to ensure that a sufficient quantity of material has been transferred. Because the proteins may retain or regain part of their structure during blotting they may react with specific antibodies giving rise to the term

immunoblotting The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detecti ...

. Alternatively the proteins may react with

s like

s giving rise to the term

affinity Affinity may refer to: Commerce, finance and law * Affinity (law), kinship by marriage * Affinity analysis, a market research and business management technique * Affinity Credit Union, a Saskatchewan-based credit union * Affinity Equity Part ...

blotting.

References

External links

* Protein (Western) Blotting - Introduction to Antibodie
Membrane Transfer
* Detailed electroblotting to PVDF procedure

{{Molecular probes Protein methods Molecular biology Electrophoresis ca:Western blot de:Western Blot es:Western blot eo:Proteina trimoo fr:Western blot it:Western blot hu:Western blot ja:ウェスタンブロッティング pl:Western-blot pt:Western blot sv:Western blot tr:Western blot ur:لطخۂ مغرب zh:蛋白質轉漬法

Development

Electroblotting procedure

See also

References

External links