Characteristics of a Useful Assay

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Characteristics of a Useful Assay
Fast Easy Sensitive
Accurate Precise Free of Interference
Pierce Protein Assay Technical Handbook, 1999
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Protein Determination
Why Quantify Protein?
It is often the denominator for presenting enzyme
activity (i.e., mol of activity per minute per mg
protein). Important to know for feeding livestock.
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Protein Determination
Biuret Reaction In alkaline solutions, Cu2
complexes with the C-N bonds in protein. The
result is a purple color. This method in
relatively insensitive. Tris buffer and other
substances often interfere.
Wharton and McCarty, 1980
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Protein Determination
The BCA (Bioinchoninic Acid) Method Uses a
similar principle as that described in the biuret
reaction except that BCA is included and
sensitivity is increased.
This process is a two-step reaction. Protein
Cu2 OH- Cu1 Cu1 2 BCA
Cu1/BCA chromophore (562 nm).
Pierce Protein Assay Technical Handbook, 1999
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Protein Determination
Lowry Method This method relies upon both the
biuret reaction and the reduction of
arsenomolybdate reagent (Folin reagent) by
tryptophan and tyrosine. Consequently, what type
of proteins will give higher absorbances? Tris
buffer and reducing compounds often interfere.
Wharton and McCarty, 1980
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Protein Determination
Warburg-Christian Method This method is a direct
spectrophotometric method. Measures absorbance
at 280 nm. This method is also very sensitive to
tyrosine and tryptophan. When might one use this
method? Absorbance at 260 nm also used to correct
for nucleic acid concentration.
Wharton and McCarty, 1980
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Protein Determination
Coomassie Blue Dye Method (a.k.a. The Bradford
Method) This method relies on the binding of
protein to Coomassie Brilliant Blue G-250 which
causes an absorbance shift from 465 nm to 595 nm.
Pierce Protein Assay Technical Handbook, 1999
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Protein Determination
Coomassie Blue Dye Method (a.k.a. The Bradford
Method) The Coomassie dye binds primarily with
basic and aromatic side chains. The interaction
with arginine is very strong and less strong with
histidine, lysine, tyrosine, tryptophan, and
phenylalanine. About 1.5 to 3 molecules of dye
bind per positive charge on the protein.
Pierce Protein Assay Technical Handbook, 1999
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Other Nitrogen Analyses
Inorganic Nitrogen Nitrate - Often determined by
converting NO3- to NO2-. Ion specific
electrodes are available. Ammonium - Several
colorimetric methods are used. Ion specific
electrodes are also an option. Because of the
ammonium/ammonia equilibrium pH is kept low to
prevent volatilization.
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Crude Protein and Total N
In feeds and other plant tissue, protein is often
calculated from the total N concentration. To
convert total N to protein, multiply by 6.25. To
convert protein to total N multiply by 0.16.
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The Kjeldahl Method
Tissue is digested in sulfuric acid to convert
nitrogen to ammonium. Ammonium is converted to
ammonia which leaves the vessel. Ammonia vapors
are then trapped. Ammonia is converted back to
ammonium for colorimetry or titration.
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The Combustion Method
Nitrogen is released after combustion at high
temperature in pure oxygen. Nitrogen is measured
by thermal conductivity detection. This
procedure is relatively expensive because of
instrument costs and pure gases.
http//www.uwex.edu/ces/forage/nfta/comb.htm
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The Combustion Method
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Near-Infrared Reflectance
A ground sample is exposed to light within a
chamber and the reflectance is measured with a
near-infrared spectrophotometer (generally 1100
to 2500 nm). Instrument is part of a system
that has been calibrated using representative
samples from population to be tested.
http//www.uwex.edu/ces/forage/nfta/comb.htm
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Bruker NIR Spectrophotometer