Electroblotting
Encyclopedia
Electroblotting is a method in molecular biology
/biochemistry
/immunogenetics
to transfer proteins or nucleic acids onto a membrane by using PVDF or nitrocellulose
, after gel electrophoresis
. The protein or nucleic acid can then be further analyzed using probes such as specific antibodies, ligand
s like lectin
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.
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."
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 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. Alternatively the proteins may react with ligand
s like lectin
s giving rise to the term affinity blotting.
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
/biochemistry
Biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...
/immunogenetics
Immunogenetics
Immunogenetics is the branch of medical research that explores the relationship between the immune system and genetics.Autoimmune diseases, such as type 1 diabetes, are complex genetic traits which result from defects in the immune system. Identification of genes defining the immune defects may...
to transfer proteins or nucleic acids onto a membrane by using PVDF or nitrocellulose
Nitrocellulose
Nitrocellulose is a highly flammable compound formed by nitrating cellulose through exposure to nitric acid or another powerful nitrating agent. When used as a propellant or low-order explosive, it is also known as guncotton...
, after gel electrophoresis
Gel electrophoresis
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...
. The protein or nucleic acid can then be further analyzed using probes such as specific antibodies, ligand
Ligand
In coordination chemistry, a ligand is an ion or molecule that binds to a central metal atom to form a coordination complex. The bonding between metal and ligand generally involves formal donation of one or more of the ligand's electron pairs. The nature of metal-ligand bonding can range from...
s like lectin
Lectin
Lectins are sugar-binding proteins that are highly specific for their sugar moieties. They play a role in biological recognition phenomena involving cells and proteins. For example, some viruses use lectins to attach themselves to the cells of the host organism during infection...
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.
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
Typically the electrophoresis gel is stained with Coomassie Brilliant Blue 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. Alternatively the proteins may react with ligand
Ligand
In coordination chemistry, a ligand is an ion or molecule that binds to a central metal atom to form a coordination complex. The bonding between metal and ligand generally involves formal donation of one or more of the ligand's electron pairs. The nature of metal-ligand bonding can range from...
s like lectin
Lectin
Lectins are sugar-binding proteins that are highly specific for their sugar moieties. They play a role in biological recognition phenomena involving cells and proteins. For example, some viruses use lectins to attach themselves to the cells of the host organism during infection...
s giving rise to the term affinity blotting.
Notations
- Protein (Western) Blotting - Introduction to Antibodies Membrane Transfer
Related links
- Detailed electroblotting to PVDF procedure - Protein Chemistry Laboratory