Western blotting
The 8 basic steps of the science of Western blotting have remained the same for the last 40 years;
- First, sample preparation: lysing and denaturing the proteins
- Next, gel electrophoresis.
- Then membrane transfer.
- After that blocking.
- Then incubating the primary antibody.
- And, then incubating the secondary antibody.
- Next detection
- And, finally analysis.
But, while the steps haven’t changed, new methods of automation for the blocking, washing and incubating the primary and secondary antibodies have increased reliability and reproducibility in Western blot research.
An Unexpected Factor That Causes Higher Background in Western Blots
Many factors can contribute to high background in WB, which include but not limited to improperly diluted antibodies, insufficient blocking, interference from blocking reagents, insufficient washing and poor quality of transfer membranes.
In addition to above mentioned factors another factor that affects the background in WB is the method/reagent used for protein extraction/sample preparation. When trouble shoots for high background in WB few researchers realize that the methods for protein extraction can significantly impact the background...
Download Invent Biotechnologie's ---> WB backgrond tip
