V. Lab | 76. SDS-polyacrylamide gel electrophoresis and Western blot

Question 1

I. SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS
1. What is Electrophoresis?

Answer 1

• Electrophoresis is a technique in which charged macromolecules (RNA, DNA, proteins, carbohydrates) are separated according to their physical properties.
• Separation is based on the fact that the charged components of a solution migrate at different velocity under the action of an electric field.

Question 2

I. SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS
2. What are used in SDS-Page?

Answer 2

• For SDS-polyacrylamide gel, polyacrylamide is used.
• SDS (sodium dodecyl sulfate) is used as a detergent that disrupts the secondary interactions of native proteins.

Question 3

I. SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS
3. What are the 2 important functions of SDS-Page?

Answer 3

1) Disrupt protein interactions + keep the denatured protein in a soluble state
2) Bind to unfolded proteins in large excess, providing extra negative charges to the molecules => consequence: each protein has roughly the same charges

Question 4

I. SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS
4. How is the mobility of different proteins during electrophoresis determined?

Answer 4

The mobility of different proteins during electrophoresis is determined mostly by the sieving effect of the porous gel:
- Smaller molecules  smaller resistance  larger mobilitylonger traveling distance
- Larger moleculelarger resistanceless mobilityless traveling distance
=> The SDS-PAGE separates proteins according
to their molecular mass (size)

Question 5

I. SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS
5. How is electric field participated in SDS-page?

Answer 5

• Essentially, an electric field is applied and the charged molecules will move through the gel.
• Since the effect of charge is eliminated by SDS and the voltage is considered constant across the gel, the molecules with smaller molecular weight will move faster and larger ones move slower.
• The bands are then compared to a reference with known molecular weight, so that the weight of the macromolecules can be estimated.

Question 6

II. WESTERN BLOT
1. What is the role of WESTERN BLOTTING?

Answer 6

Western blotting is used to detect specific proteins in a sample of tissue extract

Question 7

II. WESTERN BLOT
2. What are the steps of WESTERN BLOT?

Answer 7

1. After electrophoresis is complete, a so-called ‘’sandwich’’ is assembled in which a nitrocellulose membrane is laid tightly on the gel between filter papers and another electrophoresis is performed in a direction perpendicular to the plane of the gel in a special apparatus.
2. Proteins will be transferred from the gel onto the membrane, which makes it suitable for labeling with antibodies.
3. Detection is achieved by binding antibodies to the proteins immobilized on the nitrocellulose membrane.
4. The transferred protein is labeled with a combination of antibodies: one antibody specific to the protein of interest (primary antibody) and another antibody specific to the host species of the primary antibody (secondary antibody).
5. Secondary antibody is often complexed with an enzyme, which when combined with an appropriate substrate, will produce a detectable signal.

Question 8

II. WESTERN BLOT
3. How is detection in Western blotting achieved?

Answer 8

• Detection is achieved by binding antibodies to the proteins immobilized on the nitrocellulose membrane.
• The transferred protein is labeled with a combination of antibodies: one antibody specific to the protein of interest (primary antibody) and another antibody specific to the host species of the primary antibody (secondary antibody).
• Secondary antibody is often complexed with an enzyme, which when combined with an appropriate substrate, will produce a detectable signal.