Blotting

Question 1

Define the purpose of the following Blotting techniques:

• Northern Blotting
• Southern Blotting
• Western Blotting

Answer 1

• Northern Blotting: To transfer RNA from gel onto a carrier
• Southern Blotting: To transfer DNA from gel onto a carrier
• Western Blotting: To transfer proteins from gel onto a carrier.

Question 2

What are the steps in southern blotting?

Answer 2

• Digest DNA using restriction enzymes
• Separation of DNA via gel electrophoresis.
• Blotting: (Denature double-stranded DNA by incubation with NaOH)
• Probing: Attach transferred DNA to membrane by high temperature or UV, and incubate with a specific probe.
• Detect probe.

Question 3

What are the steps in northern blotting?

Answer 3

(ISTIA)

• Isolate RNA and treat with formaldehyde.
• Separate RNA using gel electrophoresis.
• Transfer RNA to a sheet of nitrocellulose blotting paper.
• Incubate the blot with a probe which is a labelled, single stranded DNA to form RNA-DNA molecule.
• Analyze the RNA via autoradiography.

Question 4

What are the steps in western blotting?

Answer 4

• Sample Preparation: Lysis of cells to liberate their contents.
• Gel electrophoresis using SDS PAGE and B-mercaptoethanol
• Blotting: Wet transfer for large proteins, semi-dry for small proteins.
• Probing with antibody: Probe with primary non labelled antibody, then a labelled secondary antibody.
• Detection with either colorimetric or chemiluminescent substrates.

Question 5

In western blotting, SDS PAGE is used to separate proteins. What are the function of the following chemicals in SDS PAGE?

• B Mercaptoethanol
• SDS
• Glycerol
• Bromophenol Blue.

Answer 5

• B-Mercaptoethanol: To reduce the disulfide bonds of protein.
• SDS: Disrupts the secondary and tertiary structure of protein (linearize the protein)
• Glycerol: Adds density to the protein so that it sinks in the sample well
• Bromophenol Blue: Visualize the progress of migration.