protein expression assessment by Western blot

Introduction

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 of the important stages for western blotting is the preparation of lysed samples from cells or tissues. During the lysis of cells and tissues, it should be noted that the whole process is performed on ice, and even the temperature of buffers used should be 4 ° C.

To lysate cells or tissues, a RIPA buffer mixture may be used with fresh protease inhibitor and incubated for 1 hour in ice or 4 ° C.

After centrifugation, the supernatant can be kept at a temperature of -20 to -80 ° C for a long time.

Related posts: Polyacrylamide gel electrophoresis in western blot technique – Types of materials and equipment required in Western blot techniques