Question

A. Describe a Southern blot

B. Describe a Northern blot

C. Describe a Western Blot

Just need a general consensus.

Answer 1

a. Southern blot: Uses for detection of a specific DNA sequence. This is discovered by Edwin Southern.

Steps involved in southern blotting:

1 DNA digestion - Restriction digestion helps in small frangemens sysntesis.
2 Gel electrophoresis - It helps in seperation of digest DNA
3 Blotting - Transfer of DNA fragments from Gel to Nitrocellulose paper
4 Probe labeling - helps in the identification of targatted DNA molecules.
5 Hybridization & washing - To bind at a specific sequence and washing helps in removal of unbounded probes.
6 Detection - Detection of particular DNA fragements

b.  Northern blot: Uses in gene expression studies by detection of RNA. It was developed by James Alwine, David Kemp, and George Stark.

Steps involved in Northern blotting:

1 RNA isolation - With the help of oligo (dT) cellulose chromatography mRNA can be isolated.
2 Gel electrophoresis - It helps in seperation of RNA molecules
3 Blotting - Transfer of RNA fragments from Gel to Nitrocellulose paper
4 Probe labeling - helps in the identification of targatted RNA molecules.
5 Hybridization & washing - To bind at a specific sequence and washing helps in removal of unbounded probes.
6 Detection - Detection of particular RNA fragements

c. Western blot: Useful to detect specific proteins. This was developed by Harry Towbin.

Steps involved in Western blotting:

1 Protein extraction - To avoid all other molecules, by using proper buffers protein extracted from tissues.
2 Gel electrophoresis - It helps in seperation of protein on the basis of molecular weight.
3 Blotting - Transfer of proteins from Gel to Nitrocellulose paper/ PVDF
4 Blocking - this step prevents the interactions between the membrane and the antibody used for detection of the target protein
5 Hybridization with antibodies - It binds to specific proteins.
6 Detection - Detection of particular protein molecules.