weston blocking I think will be the 10 marker.

Q: What is Western blotting?

Western blotting is a laboratory technique used to detect and analyze specific proteins in a sample using gel electrophoresis, membrane transfer, and antibody-based detection.

What are some advantages of Western blotting?

It is highly specific due to antibody binding, allows protein size estimation, and can semi-quantitatively measure protein expression levels.

What is the first step in Western blotting?

Protein extraction – Cells or tissues are lysed to extract total protein. step 1

What step follows protein extraction in Western blotting?

Protein quantification – Protein concentration is measured, often using a Bradford or BCA assay, to ensure equal loading.

What is SDS-PAGE, and why is it used in Western blotting?

SDS-PAGE separates proteins by size using a gel and electric current. SDS denatures proteins and gives them a uniform negative charge.

What happens after proteins are separated by SDS-PAGE?

Transfer – Proteins are transferred from the gel onto a membrane (usually PVDF or nitrocellulose).

What is the purpose of blocking the membrane in Western blotting?

Blocking prevents nonspecific binding of antibodies by covering the membrane with a protein solution like BSA or milk.

What is the primary antibody used for in Western blotting?

It specifically binds to the target protein of interest.

What is the role of the secondary antibody in Western blotting?

It binds to the primary antibody and is usually linked to an enzyme (like HRP) for detection.

How is the protein visualized in Western blotting?

Using a detection method (e.g., chemiluminescence), the signal from the enzyme on the secondary antibody is captured.

Why is it important to include a loading control in Western blotting?

To ensure equal protein loading across samples (e.g., using actin or GAPDH).