post lab discussion: exp. 2

Question 1

most versatile macromolecules

Answer 1

protein

Question 2

measure the concentration of total protein in a sample

Answer 2

the Bradford protein assay

Question 3

measure the concentration through measuring the spectral absorbance of samples subjected to a spectrophotometer at a specific wavelength

Answer 3

a colorimetric assay

Question 4

what is the principle behind the bradford assay

Answer 4

under acidic conditions, the coomassie dye binds to protein
during binding, the color change from brown to blue
the more protein, the more dye it binds = bluer

Question 5

what do the bradford reagent contain

Answer 5

coomassie brilliant blue G-250 (CBBG) dye
consist of:
2 sulfonic acid groups
6 phenyl groups
tht interact w the protein
+vely charged residues and hydrophobic residues

Question 6

what is the absorbance of CBBG when it forms a complex with the protein

Answer 6

465 nm to 595nm

Question 7

what are the advantages and disadvantages of the bradford protein assay

Answer 7

+ve:
quick, inexpensive, simple
compatible with the Bradford assay
measure high molecular weight proteins

-ve:
incompatible with detergents
depends on the protein sequence

Question 8

it is most commonly used protein standard due to its sensitivity to the assay than other proteins

Answer 8

BSA bovine serum albumin

it means that it react well with C. dye = reliable for accurate results

Question 9

commonly used to determine the quantity of other proteins, by comparing an unknown quantity of protein to known amounts of BSA in, for instance, the Bradford Protein Assay

Answer 9

BSA bovine serum albumin
compare - the color change

Question 10

methodology of the bradford assay

Answer 10

preparation of standard soln.
(with known amt of BSA)
- via serial dilution method
C1V1 = C2V2
*decreasing concentration

addition of bradford reagent and incubation at RT (5min)
BR - contains C. dye n added to SS and unknown sample
5min - so that the color can fully develop

reads abs at 595nm (using spectrophotometer)

linear regression (create a standard curve)

Question 11

uses a light absorbed, transmitted or reflected by the sample at each wavelength

Answer 11

US-visible (US-Vis) spectrophotometers
*measures the light absorbed, transmitted or reflected by the sample at each wavelength

Question 12

small rectangular tube to hold aqueous samples for spectrophotometric measurements

Answer 12

cuvette
*plastic, glass or quartz

Question 13

cuvette
frosted side:
transparent side:

Answer 13

frosted side: handle w/o dirtying the optical surfaces of the 2 measurement side

transparent side: allow light to pass through it

Question 14

how do u perform spectrophtmetric measurments

Answer 14

insert the cuvette in chamber
transparent side are in the east-west direction, whr the light is cming from

Question 15

reference and sample compartments contain just the solvent (blank)

Answer 15

Blank run or auto-zeroing

after auto zeroing, take out the sample cuvette, discard the soln n place the sample

Question 16

standard curve
x - axis:
y - axis:

Answer 16

x - axis: conc (mg/ml)
y - axis: absorbance (595nm)

Question 17

shows how well the plotted regression line fits the actual data

Answer 17

r-squared value (coefficient of determination)