Bradford Flashcards
Through which technique does a Bradford assay determine protein concentration?
Colorimetric spectrophotometry
Why dye is used in the Bradford assay?
Coomassie Brilliant Blue G-250
How does this dye help quantify protein levels?
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
Which compound may affect the results of a Bradford assay?
Detergents eg SDS
It interferes with the dye-protein complex
What are the advantages of a Bradford assay?
Can mostly be done just at a bench in room temp
What are the disadvantages of a Bradford assay?
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
What is PCR?
A method of copying and reproducing segments of DNA in a lab
What are the clinical applications of PCR?
Fingerprinting, genetic diagnosis, viral detection
What are the 3 main stages of PCR?
Denaturing
Annealing and cooling
Extension/elongation
Explain the denaturing phase of DNA
Sample is heated to 95 degrees to denature proteins so that the dsDNA can separate into ssDNA
Explain the second stage of PCR
The sample is cooled to its annealing temperature (which is different for every protein)
Primers then bind to the ssDNA
What is the DNA template in PCR?
The sample DNA which contains the target sequence
What are the primers in PCR?
Short pieces of ssDNA complementary to the target sequence
What is taq polymerase?
A DNA polymerase enzyme which synthesises new strands
Why are taq and Pfu polyermase used in PCR?
They are heat resistant
They can generate new strands of DNA
Describe the 3rd stage of PCR
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
What are the advantages of PCR?
Fast, cheap, automated in machine
What is the machine called that PCR takes place in?
Thermocycler
Why are 3 primers used in the PCR?
1 to bind to the WT and mutant
2 to bind to just the mutant
3 to bind just to the WT
At which end does primer 1 bind?
3’
At which end does primer 2 bind?
5’
What is sybr safe?
A dye which binds to dye
Highly sensitive for visualisation of DNA in agarose gel
How does the DNA-dye complex become visual in PCR in the gel?
It absorbs blue or UV light to become excited and visible
Why is more than 10mcg not usually used in the Bradford?
If you’re testing for something which may increase the protein conc, too big a band will be blurry and difficult to quantify
Extension times need to be longer for shorter DNAs
T/F
F
Longer extension time = longer protein
What does the sybr safe do?
Cools the gel
So it doesn’t degrade the protein
And lets you visualise the protein
