KH6

Question 1

What are some physical and chemical properties used to separate proteins?

Answer 1

Mass/Size, density, electrical charge and binding affinity.

Question 2

Describe centrifugation

Answer 2

In a spinning centrifuge, the sample receives a centrifugal force (in g) that pushes down particles. (Denser = lower in the tube)

Question 3

What affects the rate at which the pellet is formed during centrifugation?

Answer 3

Size/mass of the particle (assuming similar shapes) [Svedburg (S)]

Question 4

How can you separate multiple particles from the same sample using centrifugation?

Answer 4

Through differential centrifugation: start at lower speeds to separate heavier particles and speed up to get lighter ones.

Question 5

How can you identify density of a sample using centrifugation?

Answer 5

Through equilibrium density gradient centrifugation: gradient of high (bottom) to low (top) sucrose densities. Add the sample and spin. The sample can’t go lower than whatever density it has.

Question 6

Describe electrophoresis

Answer 6

Using an electric field, particles can migrate towards the anode or cathode based on their charge. Their speed takes into account the charge/mass ratio.

Question 7

What’s faster in gel electrophoresis: larger or smaller molecules? Why?

Answer 7

Smaller ones. The gel has this mesh structure that ultimately impedes big molecules more.

Question 8

Is it possible to only consider mass in an electrophoresis? Is there a technique that ensures this?

Answer 8

All particles would need the same charge. “SDS” is a detergent that denatures proteins (hydrophobic interactions) and coats them: uniform layer of negative charge.

Question 9

How is it efficient to use SDS-PAGE to separate proteins by mass.

Answer 9

No influence of shape and charge. Would have the same speed in a free solution.

Question 10

Does phosphorylation have an effect on SDS-PAGE results?

Answer 10

Yes. It’s believed that the phosphate group tempers with the binding of SDS. (Expected and experimental results are different)

Question 11

What’s the isoelectric point (pI)?

Answer 11

The pH at which the sum of charges in a protein is neutral (unique to each).

Question 12

What’s the effect of pH on protein charge?

Answer 12

Acidic pH favors positivity and basic pH favors negativity.

Question 13

Explain the theory of isoelectric focusing

Answer 13

Proteins have a given charge. However, there must be a certain pH at which that charge goes to 0. Amino acid composition decides what that pH is.

Question 14

How does isoelectric focusing work?

Answer 14

There’s a pH gradient in a gel. Due to an electric field, proteins move along that gradient until their charge goes neutral.

Question 15

What’s two-dimensional gel electrophoresis?

Answer 15

Combination of SDS-PAGE and isoelectric focusing.

Question 16

How does mass spectroscopy work?

Answer 16

The studied protein is ionized and accelerated in a magnetic or electric field. The acceleration gives away the m/z of the fragments.

Question 17

True or False? “There’s a relationship between pI and molecular weight”

Answer 17

False

Question 18

What’s a common way to ionize molecules?

Answer 18

Electrospray ionization

Question 19

True or False? “Just like their fragments, whole proteins can also be studied”

Answer 19

False. Too big.

Question 20

Is it useful to do MS/MS (tandem MS)?

Answer 20

Yes. The MS of the fragments gives information about the original molecule (fingerprint). Through computational analysis, we can identify the amino acid sequence.

Question 21

True or False? “Only singular amino acids are recovered by MS”

Answer 21

False. The fragmentation is partial and essentially random (some cuts are more frequent than other).

Question 22

What are proteomics?

Answer 22

The analysis of biological protein samples by MS and bioinformatics (data in computers) to identify proteins.