Question

Explanation of SDS-PAGE for Western blotting procedure, PLEASE answer the questions BELOW:

1. What does a gel electrophoresis allow you to do?

2. What is a gel?

3. How do you make the protein move, and why does this work?

4. Which protein fragments travel the furthest and why?

5. Name 3 materials used to make a gel.

6. What is polyacrylamide?

7. What is the purpose of the buffer?

8. What is the comb (in the gel) for?

9. Why do we use a protein size standard?

10. What happens to the protein once the power supply is turned on?

Answer 1

1. Gel electrophoresis allows us to seperate the different proteins or peptides present in a mixture of proteins according to their molecular weight or mass.

2.A gel is a solid- liquid mixture here the molecules are crosslinked with each other and the liquid component is dispersed in it the semi solid structure of the gel.

3.Proteins by themselves are charge molecules, in SDS PAGE, the proteins aretreated ith Sodium do-decyl sulphate an anionic reagent that gives the protein a uniform negative charge over its surface. Greater the size of the protein greater the charge over it. When the gel is placed in an electrophoresis chamber hich has a cathode and an anode and contains a suitable buffer for conduction of electrons when connected to a power supply, the proteins covered with the negative charge move through the gel towards the anode.

4.The smallest fragment moves the farthest as the gel contains pores through which the proteins pass. The smaller fragments can make their way through the pores faster than the heavier/ longer fragments.

5.3 main materials used to make a gel used in SDS PAGE are Acrylamide , bis-acrylamide, buffer. Other materials include TEMED, APS which catalyse the polymerisation of acrylamide and bis acrylamide.

6. Polyacrylamide is the water absorbent gel formed by the polymerisation of acrylamide and crosslinking with bis-acrylamide.

7.The buffers used to prepare the gel are required to maintain different pH conditions in the stacking and separating gel. The buffer used in the chamber is essential for the conduction of electricity hich drives the movement of the pprotein through the gel. The buffer also contains glycine and chloride ions which help in the stacking of the proteins according to their mass before they move into the separating gel.

8. The comb in the gel is a mould that is used to make wells in the gel. The samples are then loaded into these wells. The comb helps in making multiple wells , hich facilitate the loading of multile samles simultaneously without mixing.

9. A protein size standard consists of known proteins of different molecular weights. This is run in one of the lanes beside the samples. The proteins in the standard also move upto different distances through the gel. The bands in the samples can then be correlated ith that of the standards to determine the molecular eight of the unknon protein in the sample as same size proteins travel upto the same distance on the gel.

10.When the power supply is turned on the negatively charged proteins start migrating towards the anode.The proteins are initially stacked according to their sie in the stacking gel, they then move toards the positively charged electrode according to their sizes.