SFP4 - Protein gel electrophoresis + Western blotting

Question 1

What absorbance is relative to the concentration of proteins?

Answer 1

A280

Question 2

What is the purpose of SDS page?

Answer 2

Separates proteins based on their molecular weight

Question 3

First step in SDS page?

Answer 3

Protein samples are first denatured (unfolded)

Question 4

Second step +why?

Answer 4

Treated with SDS (sodium dodecyl sulphate) to bind and give proteins overall negative charge

Question 5

Third step?

Answer 5

Apply voltage, electrophorese through a polyacrylamide gel - effectively sieves the proteins and separates them

Question 6

Small proteins will move faster and they will be attracted to the anode (+)

Question 7

What bonds do reducing agents break?

Answer 7

Sulphide bonds

Question 8

What kind of substances are required to unfold/denature the proteins before SDS?

Answer 8

Urea, SDS reducing agent

Question 9

What type of interaction does urea disrupt?

Answer 9

Non-covalent

Question 10

What does B-mercaptoethanol act as?

Answer 10

Reducing agent

Question 11

What is the role of western blotting?

Answer 11

Identification of proteins after SDS page

Question 12

Which type of staining is more sensitive, silver or coomassie blue?

Answer 12

Silver. It is used when analysing low-abundance proteins. Spot intensity related to protein abundance.

Question 13

What are the proteins from the gel then placed onto?

Answer 13

Nitrocellulose membrane

Question 14

What does western blotting rely upon?

Answer 14

Antibodies which are highly specific to the protein of interest. Secondary antibodies can be used for detection.

Question 15

What can the secondary antibody have on it to make it detected?

Answer 15

The enzyme-conjugated secondary antibody can have an enzyme substrate which can be used as a detection signal

Question 16

What are the two steps involved in 2D gel electrophoresis?

Answer 16

1st dimension isoelectric focussing (IEF) and SDS page.

Question 17

How does IEF work?

Answer 17

Separation of proteins based on their isoelectric point (pI). A pH is established in the gel and proteins move to move to the region of the gel where the pH matches their pI. At that pH, the proteins have a net charge of zero.

Question 18

The second step of 2D gel electrophoresis?

Answer 18

SDS page - denature proteins using urea or a reducing agent. Separate proteins based on their size / denatured molecular weight.

Question 19

Up to how many different proteins are present in a cell?

Answer 19

30,000. Cells have different proteomes, giving each cell specificity.

Question 20

What is expression proteomics?

Answer 20

Analysis of expression levels of proteins in an organism

Question 21

What is a disease biomarker and what can it be used for?

Answer 21

It is a measurable substance or characteristic that is indicative of a particular disease or condition. It can be used for diagnosis, and inform about the disease mechanism (cellular pathways).