Week 5/6 weston and ELSI stages and methord

What is the first step of Western Blotting?

Protein extraction

• Lyse cells/tissue with buffer containing detergents and protease inhibitors.

• Quantify protein concentration (e.g., Bradford assay).

What happens after protein extraction in Western Blotting?

Gel electrophoresis (SDS-PAGE)

• Proteins are loaded onto a polyacrylamide gel.

• SDS gives proteins uniform negative charge → separates them by size.

What is the third step in Western Blotting?

Transfer to membrane

• Proteins are moved from the gel to a membrane (nitrocellulose or PVDF) using electric current.

• Membrane holds proteins in the same pattern.

What comes after transfer in Western Blotting?

Blocking and antibody incubation

• Block the membrane with BSA/milk to prevent non-specific binding.

• Add primary antibody (binds protein of interest), then secondary antibody (enzyme-linked).

What is the final step of Western Blotting?

Detection and analysis

• Add substrate for enzyme on secondary antibody → signal (e.g., chemiluminescence).

• Visualise using imaging → Band size and intensity reveal protein identity and quantity.

What is the first step of ELISA?

Coating the plate

• Wells of a microplate are coated with a specific antigen or antibody.

• Incubation allows binding to the plate surface

What is the second step of ELISA?

Blocking the plate

• Add a blocking buffer (e.g., BSA) to prevent non-specific binding.

• Ensures signal only comes from specific reactions.

What is the third step of ELISA?

Sample and detection antibody binding

• Add sample (e.g., serum).

• Add enzyme-linked detection antibody that binds to target antigen or antibody.

What is the fourth step of ELISA?

Substrate addition

• Add a substrate (e.g., TMB for HRP enzyme).

• Enzyme reacts with substrate → color change forms if target is present.

What is the final step of ELISA?

Detection and interpretation

• Measure color intensity with a spectrophotometer.

• Intensity of color = amount of target antigen/antibody → gives quantitative or qualitative result.