[25] Southern, Northern, and Western Blotting

Question 1

What is blotting?

Answer 1

A technique used in molecular biology to detect specific molecules in a sample.

Question 2

What is Southern blotting used for?

Answer 2

To detect a specific DNA sequence in a DNA sample.

Question 3

What is Northern blotting used for?

Answer 3

To study gene expression by detection of RNA (or isolated mRNA) in a sample.

Question 4

What is Western blotting used for?

Answer 4

To detect specific protein levels in a sample.

Question 5

What are the steps involved in Southern blotting?

Answer 5

• DNA fragmentation
• Gel electrophoresis
• Transfer to a membrane
• Hybridization with a labeled probe
• Detection

Question 6

What are the steps involved in Northern blotting?

Answer 6

• RNA extraction
• Gel electrophoresis
• Transfer to a membrane
• Hybridization with a labeled probe
• Detection

Question 7

What are the steps involved in Western blotting?

Answer 7

• Protein extraction
• Gel electrophoresis
• Transfer to a membrane
• Incubation with a primary antibody
• Incubation with a secondary antibody
• Detection

Question 8

What is gel electrophoresis?

Answer 8

A method for separation and analysis of macromolecules and their fragments, based on their size and charge.

Question 9

What is a labeled probe in the context of blotting?

Answer 9

A fragment of DNA or RNA of variable length (usually 100-1000 bases long) which is radioactively labeled and used to locate a specific sequence of DNA or RNA.

Question 10

What are primary and secondary antibodies in the context of Western blotting?

Answer 10

Primary antibody specifically binds to the protein of interest, while the secondary antibody, which carries a detectable enzyme, binds to the primary antibody.

Question 11

Who invented the Southern blot technique?

Answer 11

Edwin Southern, a molecular biologist.

Question 12

Why is it called Southern blotting?

Answer 12

It’s named after Edwin Southern, who developed this technique.

Question 13

Why are Northern and Western blotting named as they are?

Answer 13

They are named humorously after the Southern blot, which was named for its inventor.

Question 14

What is the purpose of transfer to a membrane in blotting techniques?

Answer 14

To provide a stable and robust surface where the DNA, RNA, or protein can be further analyzed.

Question 15

What kind of samples can be analyzed using these blotting techniques?

Answer 15

Biological samples, such as tissue extracts or cell lysates.

Question 16

How does gel electrophoresis separate DNA, RNA, or proteins?

Answer 16

Based on their size and charge. Smaller or more negatively charged molecules move faster through the gel.

Question 17

What is hybridization in the context of Southern and Northern blotting?

Answer 17

The process where the labeled probe binds to its complementary sequence in the sample.

Question 18

What is detection in the context of blotting techniques?

Answer 18

The process of visualizing the labeled probes or antibodies that have bound to their target, indicating the presence of the molecule of interest.

Question 19

What is RNA?

Answer 19

Ribonucleic acid, a polymeric molecule essential in various biological roles.

Question 20

What is DNA?

Answer 20

Deoxyribonucleic acid, the molecule that contains the genDeoxyribonucleic acid, the molecule that contains the genetic instructions for the development and functioning of living organisms.