P2 - Bradford Assay

Question 1

what is the max absorbance of bradford dye reagent?

Answer 1

595nm

Question 2

what is the max absorbance of nucleic acid?

Answer 2

260nm

Question 3

what is the A280 assay used for?

Answer 3

to roughly determine protein concentration, if it is not contaminated with DNA

Question 4

when does the A280 absorbance trick not work?

Answer 4

when DNA is present in protein assay

Question 5

how does biuret assay determine protein concentration?

Answer 5

detection of blue complex between biuret agent and peptide bond

Question 6

which assay (bradford vs biuret) is better for smaller protein concentration detection?

Answer 6

bradford

Question 7

which assay is better for detection of large protein concentrations?

Answer 7

biuret

Question 8

how does bradford measure protein concentartion?

Answer 8

by measuring concentration of aromatic residues

Question 9

how does biuret measure protein concentration?

Answer 9

by measuring amount of peptide bonds

Question 10

If 45% of the light is transmitted by a sample in cuvette, the absorbance reading is?

Answer 10

0.35

Question 11

If 60% of the light is transmitted by a sample in cuvette, the absorbance reading is

Answer 11

0.222
formula: 2-log(60)

Question 12

in SDS page which comes first running or tracking gel?

Answer 12

running gel and stacking gel on top

Question 13

what is running gel made from?

Answer 13

15% POLYacrylamide

Question 14

what is stacking gel made from?

Answer 14

7% acrylamide

Question 15

what is the function of acrylamide?

Answer 15

it is a mesh-like matrix to separate proteins based on size

Question 16

what happens SDS when it is out of solution?

Answer 16

white crystals form

Question 17

what is TEMED used for?

Answer 17

to catalyse polyacrylamide polymerisation (with APS)

Question 18

what is butanol saturated water used for?

Answer 18

to allow gel to polymerise without matrix drying out

Question 19

what is the need for sodium dodecyl sulfate? SDS

Answer 19

to eliminate charge on proteins

Question 20

what is the need for Tris-HCl?

Answer 20

buffer - pH 8.7, keeps proteins linear

Question 21

what is the need for acetic acid in SDS page?

Answer 21

provides an acidic environment - enhancing dye interaction

Question 21

what is the need for acetic acid in SDS page?

Answer 21

provides an acidic environment - enhancing dye interaction

Question 22

when is polyacrylamide considered a neurotoxin?

Answer 22

in solid and liquid form

Question 23

why is kanamycin used?

Answer 23

prevention of bacterial contamination

Question 24

what happens proteins undergoing SDS page?

Answer 24

reducing agent, ionic detergent, fractionation by electrophoresis

Question 25

what does reducing agent do?

Answer 25

breaks disulphide bonds - protein unfolding

Question 26

what does ionic detergent do in SDS page prep?

Answer 26

enables solubilisation, linearisation, uniform charge

Question 27

what is an example of an ionic detergent?

Answer 27

SDS

Question 28

what is an example of a reducing agent?

Answer 28

2-mercaptoethanol