Bradford

Question 1

Through which technique does a Bradford assay determine protein concentration?

Answer 1

Colorimetric spectrophotometry

Question 2

Why dye is used in the Bradford assay?

Answer 2

Coomassie Brilliant Blue G-250

Question 3

How does this dye help quantify protein levels?

Answer 3

Binds to the arginine and aromatic residues in the protein
This shifts the light absorbance of the dye
A standard curve is formed from this and then unknown can be plotted on this graph

Question 4

Which compound may affect the results of a Bradford assay?

Answer 4

Detergents eg SDS

It interferes with the dye-protein complex

Question 5

What are the advantages of a Bradford assay?

Answer 5

Can mostly be done just at a bench in room temp

Question 6

What are the disadvantages of a Bradford assay?

Answer 6

Proteins without a good number of arginine and aromatic resides will not bind as well
The protein concentration being measured must have similar reactions with the dye as the standard protein

Question 7

What is PCR?

Answer 7

A method of copying and reproducing segments of DNA in a lab

Question 8

What are the clinical applications of PCR?

Answer 8

Fingerprinting, genetic diagnosis, viral detection

Question 9

What are the 3 main stages of PCR?

Answer 9

Denaturing
Annealing and cooling
Extension/elongation

Question 10

Explain the denaturing phase of DNA

Answer 10

Sample is heated to 95 degrees to denature proteins so that the dsDNA can separate into ssDNA

Question 11

Explain the second stage of PCR

Answer 11

The sample is cooled to its annealing temperature (which is different for every protein)
Primers then bind to the ssDNA

Question 12

What is the DNA template in PCR?

Answer 12

The sample DNA which contains the target sequence

Question 13

What are the primers in PCR?

Answer 13

Short pieces of ssDNA complementary to the target sequence

Question 14

What is taq polymerase?

Answer 14

A DNA polymerase enzyme which synthesises new strands

Question 15

Why are taq and Pfu polyermase used in PCR?

Answer 15

They are heat resistant

They can generate new strands of DNA

Question 16

Describe the 3rd stage of PCR

Answer 16

The sample is heated back up to a lower temp then the denaturing temperature
Taq polymerase then binds to the DNA and synthesises new strands to be made

Question 17

What are the advantages of PCR?

Answer 17

Fast, cheap, automated in machine

Question 18

What is the machine called that PCR takes place in?

Answer 18

Thermocycler

Question 19

Why are 3 primers used in the PCR?

Answer 19

1 to bind to the WT and mutant
2 to bind to just the mutant
3 to bind just to the WT

Question 20

At which end does primer 1 bind?

Answer 20

3’

Question 21

At which end does primer 2 bind?

Answer 21

5’

Question 22

What is sybr safe?

Answer 22

A dye which binds to dye

Highly sensitive for visualisation of DNA in agarose gel

Question 23

How does the DNA-dye complex become visual in PCR in the gel?

Answer 23

It absorbs blue or UV light to become excited and visible

Question 24

Why is more than 10mcg not usually used in the Bradford?

Answer 24

If you’re testing for something which may increase the protein conc, too big a band will be blurry and difficult to quantify

Question 25

Extension times need to be longer for shorter DNAs

T/F

Answer 25

F

Longer extension time = longer protein

Question 26

What does the sybr safe do?

Answer 26

Cools the gel
So it doesn’t degrade the protein
And lets you visualise the protein