Activity 8

Question 1

ACTIVITY 8 TITLE

Answer 1

Protein Quantitation by Bradford Protein Assay using Spectrophotometry

Question 2

This experiment is designed for testing the ___ of common food or biologically derived material

Answer 2

Protein content

Question 3

PROTEIN QUANTITATION is often necessary before processing protein samples for ___ and ___

Answer 3

Isolation

Separation

Question 4

Types of analysis on protein samples

Answer 4

Chromatographic

Electrophoretic

Immunochemical

Question 5

Quick, simple, and inexpensive laboratory procedure to determine the PROTEIN CONTENT of a sample.

Answer 5

Bradford Protein Assay

Question 6

Measurement most commonly used in protein assays is ___

____ tells us that if a SOLUTE absorbs light of a PARTICULAR WAVELENGTH, the ABSORBANCE is DIRECTLY PROPORTIONAL to the CONCENTRATION OF THE SOLUTE in SOLUTION

Answer 6

Absorbance of Light

Beer’s Law

Question 7

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

The solute itself DOES NOT ABSORB LIGHT. So, a ___ is used to produce colored compounds in proportion to the concentration of unknown.

Answer 7

UV-Vis Spectrophotometer

Reagent

Question 8

Principle:

The ASSAY is based on the observation that the ABSORBANCE MAXIMUM for an acidic solution of ___ shifts from ___ to ___ when BINDING TO PROTEIN OCCURS

Both HYDROPHOBIC and IONIC interactions stabilize the ANIONIC form of the dye, causing a visible color change.

Answer 8

Coomassie Brilliant Blue G-250

465 nm to 595 nm

Question 9

Principle:

The ASSAY is USEFUL since the EXTINCTION COEFFICIENT of a DYE-ALBUMIN COMPLEX solution is constant over a ____.

Answer 9

10-fold concentration range

Question 10

Equipment:

In addition to standard, liquid handling supplies, a ___ is needed, with maximum transmission in the region of ___, on the border of the visible spectrum.

___ or ___ may be used, however the color reagent stains both. Disposable is recommened

Answer 10

Visible Light Spectrophotometer

595 nm

Glass or Polystyrene curvettes

Question 11

Unknown proteins in Activity 8

Answer 11

Keratin

Myoglobin

Albumin

Question 12

Reagent and standard used

Answer 12

Bradford Reagent

BSA protein standard

Question 13

How much bradford reagent do we need?

What about for each test tube?

Answer 13

60 mL

5 mL

Question 14

Test tube 1-5

Volume BSA standard solution, uL

Answer 14

Calculated BSA concentration, mg/mL
10 - 0.002

20 - 0.004

30 - 0.006

40 - 0.008

50 - 0.01

Question 15

How much unknown needed?

Answer 15

25 uL