Protein Techniques

Question 1

Why gene expression techniques unreliable when telling you about protiens even though the gene makes the protein?

Answer 1

but this is an unreliable indicator of protein expression as the number of genes does not indicate how much protein there is as they could be short lived.

Question 2

Are proteins heterogenous?

Answer 2

yes

Question 3

How do we detect proteins?

Answer 3

Probes for proteins – stains or dyes.

Probes for specific proteins – antibodies

Question 4

What does SDS-PAGE electrophoresis seperate on the basis of?

Answer 4

size

Question 5

Do you have to denature the proteins before running them through a gel electrophoresis on SDS-PAGE?

Answer 5

Yes to get individual proteins

Question 6

How do you detect protiens in SDS-PAGE?

Answer 6

stain or dye

Question 7

In a 2-D PAGE analysis what is the 1st dimension?

Answer 7

basis of charge - will flow towards end with opposite charge

Question 8

In 2-D PAGE analysis what is the 2nd dimension’s basis for splitting molecules?

Answer 8

basis of size

Question 9

How can antibodies be used to identify proteins?

Answer 9

Antibodies are raised against purified proteins or synthetic peptides (inject animal with inflammation get antibody).

It will bind to its specific protien when in its presence

Question 10

What does ponyclonal mean?

Answer 10

several antibodies to different epitopes – is finite as it is unique to that animal

Question 11

What does monoclonal mean?

Answer 11

a single antibody to a single epitope – this is limitless.

Question 12

what is western/immuno blotting?

Answer 12

Blotting separated proteins onto a nylon membrane and the antibodies will stick when they find a protein, they have an affinity to.

Question 13

How can you make the antibody bound on a western blot easier to see?

Answer 13

As antibodies have the fc domain you can get a second round of reagents which will bind specifically to the antibody which will make them easier to see as they can be fluorescent

Question 14

What is co-immunoprecipitation?

Answer 14

Antibodies will stick to a protein in the solution. You can then purify the antibody by using the bacterial proteins to purify the protein the antibody is attached to.

Question 15

What is the yeast two hybrid system?

Answer 15

Genetic screen to look for direct protein, protein interaction specifically looking at the part which bound to promoter and the part which activated the protein.

Question 16

What does mass spectrometry measure?

Answer 16

Can measure a mass and a charge.

Question 17

How do you do mass spectrometry?

Answer 17

Digest the protein we want to examine with trypsin to get peptides and then measure the base and the charge of these. You could then use a computer to find say a lysine (or any other base/amino acid).

Question 18

How can you tell what the protein is from a mass spectrometer?

Answer 18

You only need a few peptides to know what the protein is.

Can find the proteins based on the tryptic fragments you have made

Question 19

Can you tell is two proteins are close together using a mass spectrometer?

Answer 19

Yes as the tryptic bits will be stuck together

Question 20

How can you tell is a protein is interacting with another? (Surface plasmon resonance)?

Answer 20

The properties on the surface of a protein will change if they are interacting – this will give to quantative measurements.The resonance angle will change

Question 21

How does Surface plasmon resonance (SPR) occurs?

Answer 21

when polarized light strikes an electrically conducting surface at the interface between two media. This generates electron charge density waves (plasmons), reducing the intensity of reflected light at a specific angle known as the resonance angle that is proportional to the mass on a sensor surface.