Service description
One of the tasks carried out in the EEZA within the laboratory of Animal Physiology for diferenetes studies Evolutionary Ecology is the Western blot for the transfer, immobilization and detection of stress proteins from red blood cells of different bird species, among which are at least three species of penguins.
In this technique, proteins from a complex sample are separated by electrophoresis in polyacrylamide gel according to their filling properties and size as polyacrylamide gels have size close pore size of proteins and function as a molecular sieve in which we can vary the pore size by varying the gel composition.
In this type of electrophoresis proteins are subjected to migration beforehand denatured and suffer loss of their three-dimensional structure by the action of an ionic detergent sodium dodecyl sulfate, this own load blocks binding molecule and protein detergent complexes gives -protein uniform loads negative making all proteins migrate to the anode being greater or less mobile depending upon the molecular mass of the protein (a lower molecular mass greater mobility and vice versa) thus can determine the apparent molecular weight of any protein , by comparison with a known standard (molecular weight markers) as the rate of separation in these conditions is proportional to the mass of the protein.
Then, the proteins are transferred to a nitrocellulose membrane hydrophobic, the gel using a perpendicular electric field when proteins are transferred in their accessible surface to interact with other molecules that allow specific detection.
To prevent nonspecific binding and increasing the signal baseline, the membrane areas that are not covered by proteins using proteins or other molecules that are not recognized by the primary and secondary antibodies are blocked. Then the membrane is incubated with a specific primary antibody to the antigen. After several washing steps, incubated with a labeled secondary antibody directed against the primary antibody. Labelling was performed by covalent attachment of enzymes.
Finally the protein bands recognized by the antibodies are revealed after incubation with substrates that generate colored insoluble products.
Options and prices chart
Options |
Unit |
Public Sector |
Other customers |
Electroblotting |
2 geles
|
666.59 € |
730.08 € |