Question

Q/ Write and explain 2 techniques that used for gene expression analysis on protein level??

Answer 1

Two techniques that used for the gene expression analysis on protein levels are 1) Western Blot and 2) Fluorescence activated cell sorting.

1) Western Blotting: It is a widely used technique to check the expression of specific protein in tissue samples homogenate and cell extracts. In this technique specific proteins are detected by using the specific monoclonal antibody against that protein

Sample preparation and gel electrophoresis:Cells and tissues are lysed by lysis buffer and take supernatant cotaininig proteins and protein samples are denatured by applying heat and denature agents like SDS and beta mercaptoethanol.and run on polyacrylamide gel electrophoresis.Gel percentage depends on the molecular weight of protein is separated.

Protein Transfer and Immunoblotting: Protein is separated on PAGE and transfer on to the nitrocellulose membrane and PVDF membrane and after protein transfer on the membrane, membrane is stained with ponceau stain to check whether protein transfer or not.Wash the membrane to remove the ponceau stain and block the membrane with milk and BSA and stain with the primary antibody against the protein of interest for overnight at 4 degree and wash the primary antibody and add secondary antibody with tag against the primary antibody.Develope the blot for detection or to check the expression of specific protein by using substrate and chemoluminescent detection.

2) Fluorescene activated Cell sorting: In this techniques, protein expression are checked by using the fluorescence tag monoclonal antibodies against the specific protein. Cells are stained by two methods 1) Surface staining to check the expression of cell surface protein like receptors and 2) Intracellular staining to check the expression of intracellular proteins like cytokines and chemokines.

Procedure: Harvest the cells and wash to remove the growth medium on 1200rpm for 10 min at room temperature and stained the cells with surface protein specific FACS antibodies for 30 min.Wash the surface antibody and permeablised cells using lysing and perm solution. Wash the cells to remove perm solution and stained the cells with intracellular proteins antibodies for 1 hours and wash the cells with sheath or wash buffer.Acquired the cells on flow cytometery machine to check the expression of preteins.The Flow cytometry data is analysed on the software flow Jo or Diva software.