SDS PAGE TUTORIAL

Question 1

What is the purpose of the gel in gel electrophoresis?

Answer 1

Gel is used as a size-selective sieve; facilitating separation based on molecular weight

Question 2

What is the full form of SDS-PAGE?

Answer 2

Sodium Dodecyl Sulfate – PolyAcrylamide Gel Electrophoresis

Question 3

What are stacking and resolving gels and where are they found?

Answer 3

1. Stacking gel - found at the start of the gel; it promotes concentration or “stacking” of the proteins in a sample,
   Separating gel - found after the stacking gel; it separates the proteins according to size.

Question 4

What is the general process of an SDS PAGE?

Answer 4

1. Lyse the sample, and denature with buffer containing SDS and β-mercaptoethanol. Heat the sample at 95deg for 5 min
2. Load onto gel and perform gel electrophoresis for 2 hours
3. Prepare a cold transfer buffer and membrane. Make a transfer sandwich with the gel at the cathode and membrane at the anode so the proteins migrate to the membrane.
4. Immunodetection

Question 5

What is the use of SDS and β-mercaptoethanol ?

Answer 5

1. SDS denatures the proteins in the sample and binds to their polypeptide backbones, giving them a uniform negative charge.
2. β-mercaptoethanol breaks disulfide bonds in the proteins, further disrupting the protein structure and giving the polypeptides a similar elongated shape.

Question 6

What are the types of lysis buffers?

Answer 6

1. NP-40 buffer or Triton X-100-containing buffer
2. Zwitterionic detergents
3. RIPA (radioimmunoprecipitation assay) buffer, which contains SDS, is good for whole cell extracts, membrane-bound extracts, and nuclear extracts, but disrupts protein-protein interactions.

Question 7

Describe the process of wet electroblotting

Answer 7

1. Gel and membrane sandwiched together, with absorbent filter paper and sponges, and clamped into a cassette to prevent formation of air bubbles between the gel and membrane.
2. The sandwich is submerged vertically in a conducting transfer buffer and exposed to an electric field.
3. Negatively charged proteins migrate out of the gel towards the positively charged electrode and become immobilised on the membrane.

Question 8

Describe the process of semi-dry electroblotting

Answer 8

The gel and membrane are again sandwiched together with absorbent paper in a horizontal apparatus that is placed in direct contact with electrodes.

Question 9

What is the problem with wet electroblotting

Answer 9

It is a time-consuming process, typically requiring ~1 hour to overnight.

Question 10

What is wet and semi dry electroblotting suited to?

Answer 10

Wet: low to high MW proteins
Semi-dry: low MW proteins

Question 11

What are the steps of immunodetection once the proteins are transferred to the membrane?

Answer 11

1. Blocking: Prevents non-specific antibody binding.
2. Washing: Removes unbound antibodies and reduces background signal.
3. Antibody incubations: Primary antibodies recognize target epitopes, and labeled secondary antibodies enhance detection.
4. Detection: Enzyme-conjugated secondary antibodies react with a substrate, generating a signal for visualization.

Question 12

What is native PAGE?

Answer 12

1. Native PAGE is a technique that uses non-denatured gels for the separation of proteins.
2. Unlike SDS PAGE, no denaturing agent is added in the preparation of gels.
3. As a result, the separation of proteins takes place on the basis of charge and size of the proteins.