Western blotting Flashcards
(13 cards)
What is the main purpose of Western blotting?
To detect and measure a specific protein in a sample.
What does SDS-PAGE stand for?
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis.
What is the role of SDS-PAGE in Western blotting?
It separates proteins based on size (molecular weight).
What happens after proteins are separated by SDS-PAGE?
They are transferred to a membrane (blotting step).
What materials are commonly used for blotting membranes?
Nitrocellulose or PVDF.
Why is blocking performed after transfer to the membrane?
To prevent non-specific antibody binding.
What is the role of the primary antibody in Western blotting?
It binds specifically to the target protein.
What is the role of the secondary antibody?
It binds to the primary antibody and carries a label for detection.
How is the target protein visualized on a Western blot?
Through a chemiluminescent, colorimetric, or fluorescent signal.
What does the position of the band on the blot indicate?
The size of the protein.
What does the intensity (darkness) of the band indicate?
The amount of protein present.
What does ‘Western’ refer to in ‘Western blot’?
It’s a play on the names of other blots (Southern = DNA, Northern = RNA).
Can Western blotting distinguish between protein isoforms?
Yes, if they have different sizes.
