Activity 8 Flashcards
ACTIVITY 8 TITLE
Protein Quantitation by Bradford Protein Assay using Spectrophotometry
This experiment is designed for testing the ___ of common food or biologically derived material
Protein content
PROTEIN QUANTITATION is often necessary before processing protein samples for ___ and ___
Isolation
Separation
Types of analysis on protein samples
Chromatographic
Electrophoretic
Immunochemical
Quick, simple, and inexpensive laboratory procedure to determine the PROTEIN CONTENT of a sample.
Bradford Protein Assay
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
Absorbance of Light
Beer’s Law
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.
UV-Vis Spectrophotometer
Reagent
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.
Coomassie Brilliant Blue G-250
465 nm to 595 nm
Principle:
The ASSAY is USEFUL since the EXTINCTION COEFFICIENT of a DYE-ALBUMIN COMPLEX solution is constant over a ____.
10-fold concentration range
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
Visible Light Spectrophotometer
595 nm
Glass or Polystyrene curvettes
Unknown proteins in Activity 8
Keratin
Myoglobin
Albumin
Reagent and standard used
Bradford Reagent
BSA protein standard
How much bradford reagent do we need?
What about for each test tube?
60 mL
5 mL
Test tube 1-5
Volume BSA standard solution, uL
Calculated BSA concentration, mg/mL
10 - 0.002
20 - 0.004
30 - 0.006
40 - 0.008
50 - 0.01
How much unknown needed?
25 uL
