Electroblotting

Electroblotting

Electroblotting is a method in molecular biology/biochemistry/immunogenetics to transfer proteins or nucleic acids onto a membrane (generally PVDF or nitrocellulose) after gel electrophoresis. The protein or nucleic acid can then be further analyzed using probes such as specific antibodies 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.

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." [ [http://www.patentstorm.us/patents/4840714.html United State Patent 4840714] ]

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 cathode, as the current and sample will be moving in that direction (the protein will have a negative charge due to interactions with SDS). 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 Blue following the transfer to ensure that a sufficient quantity of material has been transferred.

References

Footnotes

Notations

* Information on Electroblotting [http://www.serva.de/products/sheets/blotting.shtml Serva Electrophoresis]
* Protein (Western) Blotting - Introduction to Antibodies [http://www.chemicon.com/resource/ANT101/a2B.asp#Membrane Membrane Transfer]

ee also

* Western blotting
* SDS-page
* Gel electrophoresis

Related Links

* Detailed electroblotting to PVDF procedure - [http://pcl.calabreso.com/protocols/pvdf_pro.html Protein Chemistry Laboratory]


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