Michaelmas week 3 Flashcards
What is the wavelength of max light absorption of DNA (relevant for Beer’s law)
260nm
What is a spectophotometer
Measures how much molecules absorb light
What is Beer’s law?
A=cEL
A in arbitrary units
E molar extinction coefficient in litres per cm per mol
For nucleotides how and why are exctinction coefficient units different?
Expressed in grams not moles
For linear molecules length (=weight) of the molecule contributes more than the number of moles and so c is expressed in gL-1 instead of M
What is the Bradford assay and what dye does it use?
Measures protein concentration, based on the Coomassie dye which changes colour when it binds amino acids
What do you need to do before a Bradford assay?
Zero with Bradford reagent and no protein
What colour does the cationic form of bradford reagent absorb most?
Red
What colour does the anionic form absorb most and what is A max?
Blue, A max 595nm
What does binding to a protein do to colours
In acidic environments the protonated form is most favourable
Binding to the protein favours the stability of the anionic form (dye binds to amide groups)
Draw an example standard curve and how you would read off it
(See notes for diagram)
What happens to relationship between A595 and concentration at high conc?
Stops being linear, so we can’t use beer’s law as there isn’t a standard value of E
Instead plot standard curve
What volumes do P10, P20, P200, P1000 and P5000 Gilson pipettes dispense?
2-10 microlitres 2-20 10-200 100-1000 500-5000
In a P1000, P200 and a P20 what would ‘087’ on a reading mean?
870 microlitres
87
8.7
Always use a pipette…
With max volume closest to the volume you’re trying to dispense
How do you extract a soluble protein from tabacco leaves?
Leaf extracts are first kept on ice
Then ground up, disrupting lysozymes and vacuoles
