Three Techniques to detect Proteins

Question 1

What does SDS do?

Answer 1

Separate proteins according to their size or molecular weight .

Larger the size , higher molecular weight slower migration .

Question 2

What are the two forms of PAGE

Answer 2

Native page

Remain in their native form

Separated by charge , density , size and shape

Separation and detection of enzymes

Determined by charge and size

Question 3

What does SDS do ?

Answer 3

Proteins are denatured using SDS and reducing agents

Seperated by size

Determination of molecular weights of proteins

Determined by size

Are heated at 100 degrees

B-mercaptoethanol or DTT

Proteins migrate towards positive electrode seperated by their molecular weights

Question 4

Western Blotting

Answer 4

Detection and analysis of proteins

Immunoblotting because antibody probes are used to detect target proteins or membranes

Question 5

Steps of Western Blotting

Answer 5

1. Protein gel electrophoresis - Seperation of proetins by gel electrophoresis .
2. Protein Transfer- gel electrophoresis / Transfer
3. Blocking
4. Antibody Probing
5. Detection

Question 6

Application

Answer 6

Aim to detect specific proteins in the sample .

-The western blot is routinely used for verification of protein production after cloning. It is also used in medical diagnostics, e.g., in the HIV test or BSE-Test.

Question 7

Why is Western Blotting important technique used in protein ?

Answer 7

Western blotting is an important technique used in cell and molecular biology.

By using a western blot, researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells.

The technique uses three elements to accomplish this task:

(1) separation by size,
(2) transfer to a solid support, and
(3) marking target protein using a proper primary and secondary antibody to visualize.

This paper will attempt to explain the technique and theory behind western blot, and offer some ways to troubleshoot.

Question 8

Dot blot/Slot blot

Answer 8

Technique used to detect proteins

Question 9

Uses

Answer 9

Chamotherapy / gel electrophoresis .

Question 10

Advantage

Answer 10

Performing a dot blot is similar in idea to performing a western blot, with the advantage of faster speed and lower cost.

Dot blots are also performed to screen the binding capabilities of an antibody.

Question 11

Disadvantages

Answer 11

separate proteins by size.

This can make detection of a false positive (such as non-specific binding to peptide fragments) difficult to detect.

Question 12

Advantages of Western Blot and Disadvantages of Western Blot

Answer 12

Advantages - are sensitivity and specificity.

Disadvantages - it is time-consuming

Question 13

Advantages of SDS and Disadvantages

Answer 13

A- Requires small amount of sample

D- Highly costly