ACT 8

Question 1

Act 8 Title

Answer 1

Protein Quantification by Bradford Protein Assay using Spectrophotometry

Question 2

Objectives

Answer 2

This experiment is designed for testing the protein content of common foods or any source of biologically derived material.

Question 3

is often necessary before processing protein samples for isolation, separation, and analysis by chromatographic, electrophoretic, and immunochemical methods.

Answer 3

Protein quantitation

Question 4

is a quick, simple and inexpensive laboratory procedure to determine the protein content of a sample.

Answer 4

Bradford protein assay

Question 5

the measurement most commonly used in protein assays is _____ which is proportional to the concentration of the substance of interest

Answer 5

absorbance of light

Question 6

tells us that if a solute absorbs light of a particular wavelength, the absorbance is directly proportional to the concentration of that solute in solution.

Answer 6

Beer’s law

Question 7

is used to measure, display, and/or record absorbance in quantifiable units.

Answer 7

UV-Vis Spectrophotometer

Question 8

True or False

The solute should absorb light for a practical assay.

Answer 8

False

Often the solute by itself does not absorb light so as to allow for a practical assay

Question 9

Employing one or more _____ to produce colored compounds in proportion to the concentration of an unknown is possible.

Answer 9

reagents

Question 10

The assay is based on the observation that the absorbance maximum for an acidic solution of ________________________ shifts from 465 nm to 595 nm when binding to protein.

Answer 10

Coomassie Brilliant Blue G-250

Question 11

Absorbance shift (in nm)

Answer 11

465 nm to 595 nm

Question 12

Both _______ and __________ interactions stabilize the anionic form of the dye, causing a visible color change.

Answer 12

hydrophobic and ionic interactions

Question 13

The assay is useful since the extinction coefficient of a dye-albumin complex solution is constant over a ____________ concentration range.

Answer 13

10-fold

Question 14

is needed, with maximum transmission in the region of 595 nm, on the border of the visible spectrum.

Answer 14

visible light spectrophotometer

Question 15

sample holder for spectrophotometry

Answer 15

glass or polystyrene cuvettes

Question 16

Materials (7)

Answer 16

Bradford reagent
BSA protein standard
50-ml burette
iron stand
burette clamp
(8) 10 mL test tubes
test tube rack

Question 17

wash (8) 10-ml test tubes and dry in the oven for ______

Answer 17

15 minutes

Question 18

volume of Bradford reagent in each test tube

Answer 18

5 mL

Question 19

dilution equation

Answer 19

C1V1 = C2V2

Question 20

BSA concentration (mg/mL)

Answer 20

1.00 mg/mL

Question 21

Volume BSA Standard Solution for the Calibration curve (5)

Answer 21

10, 20, 30, 40, 50 microL = 0.01 -> 0.05 ml

Question 22

Volume BSA Standard Solution for the unknowns

Answer 22

25 microL = 0.025 mL

Question 23

wavelength setting

Answer 23

595 nm

Question 24

UV-Vis meaning, which are regions of the electromagnetic spectrum

Answer 24

ultraviolet-visible

Question 25

excellent curve range

Answer 25

R2 > 0.98

Question 26

good curve range

Answer 26

0.9025 - 0.98

Question 27

fair curve range

Answer 27

0.8100 - .9025

Question 28

3 unknowns

Answer 28

Keratin
Myoglobin
Albumin