Expression of cell or tissue protein by western blot method
Western Blot is a technique used to identify a protein from a complex mixture of proteins extracted from cells and tissues.
Western Blot has three stages :
- Isolation based on the size of proteins by SDS-PAGE gel
- Transfer to the membrane
- Labeling of target proteins using primary and secondary antibodies
Tissue or Cell Lysis
◀ One ofthe important stages for western blotting is the preparation of lysed samplesfrom cells or tissues. During the lysis of cells and tissues, it should benoted that the whole process is performed on ice, and even the temperature ofbuffers used should be 4 ° C.
◀ Tolysate cells or tissues, a RIPA buffer mixture may be used with fresh proteaseinhibitor and incubated for 1 hour in ice or 4 ° C.
◀ Aftercentrifugation, the supernatant can be kept at a temperature of -20 to -80 ° Cfor a long time.