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Lab 3.
PROTEIN DETERMINATION
I.
INTRODUCTION
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You will use these assays to determine the protein concentration in an artificial
unknown and in serum.
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All assay methods have a characteristic dynamic response range. This is the
range of sample concentrations over which the assay produces a differential
response. Consider the situation shown in the graph below:
.
assay response
100%
0%
amount of protein in sample
dynresp
Specificity
I.A.3.
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Sample Recovery
In some situations the amount of sample available may be very limited. In such
cases one would hope to recover the protein from the assay so that it could be
manipulated subsequently. In this case one would want a "non-destructive"
assay. Which of the 3 assay methods presented here is non-destructive?
I.A.4.
Under the heading of logistics we include a variety of issues related to the cost
of materials and labor. You must consider the cost of equipment, chemical
reagents, and staff time to prepare materials and perform assays. Increasingly,
the cost of disposal of hazardous materials is becoming an important issue.
I can assure you that these issues were carefully considered and are a
significant constraint to the design of this laboratory exercise.
I.A.5.
Safety
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Protein assays based on the Biuret reaction were developed and used by numerous
investigators throughout the first half of this Century. Typically each investigator
adapted the basic reaction to suit their own needs, introducing their own variations of
concentrations and assay conditions. Thus there are many versions of Biuret assay
in the earlier literature.
The Biuret Assay conditions normally specified by contemporary sources are based
directly on the work of Gornall et al. (1948), usually without attribution. These
workers conducted an extensive investigation of the effect of reagent concentrations,
pH and incubation conditions to arrive at the recipes and procedures we now employ
without much question. (Likely few people performing the Biuret Assay according to
the procedure of Gornall et al. stop to ask themselves why the reagent contains
potassium tartrate.) We do well to remember that these investigators were studying
hepatic lesions, and therefore specifically optimized the assay conditions for
determining proteins in serum.
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Wait 5 minutes.
I.C. UV absorbance
Virtually all proteins exhibit a strong UV absorbance maximum near 280 nm. This
characteristic absorbance is due almost entirely to the absorbance by the aromatic
rings in the R-groups of the amino acids tryptophan and tyrosine. (As you will recall,
these are 2 of the 20 amino acids ordinarily found in proteins.)
Samples are assayed directly in a spectrophotometer capable of generating and
measuring UV light at this wavelength. No chemical reagent is involved here, as in
the Biuret assay, because the UV absorbance is an intrinsic property of the protein
itself.
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II.
PROCEDURES
We provide a standard stock solution of protein at [20 mg/ml]. The protein is bovine
serum albumin (BSA). The first task will be to dilute the stock protein solution to give
a series of solutions with different, known concentrations (Procedure II.A). This must
be done first.
The protein standard solutions will be used to construct standard curves for the 2
assay procedures (II.B, II.C). These can be completed in any order, though the
availability of UV spectrophotometers is limited. Therefore, some groups should do
the UV absorbance assay first.
In addition to the protein standard solutions prepared in II.A, there will also be a
simple "unknown" solution. The unknown is a pure solution BSA in water. Dilutions
of the unknown should be prepared at dilution factors of 10-1 and of 10-2.
This is summarized as follows:
Finally, you will determine the protein concentration in a real sample of serum.
Each pair of students will work with a reagent "Kit" containing the following:
Biuret Reagent
Bovine serum Albumin Protein Standard [20 mg/ml]
Protein Unknown
Serum
Be sure you do not confuse the standard with the unknown protein solution.
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3.8
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3.9
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3.10
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6. Liquid waste disposal: When you are satisfied that your results are correct,
discard all the assay solutions (not the standards) by pouring the contents of
each microcentrifuge tube into a waste beaker designated for Biuret Reagent.
Rinse each tube with a small amount of di water from a squeeze bottle and add
this rinse to the waste beaker. After rinsing, the empty microfuge tubes are
placed in the receptacle provided.
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II.C. UV Absorbance
In this assay we use the intrinsic ability of proteins to absorb ultraviolert (UV) light.
The UV absorbtion of native proteins is due almost entirely to the presence of the
aromatic amino acids tryptophan and tyrosine which have absorbtion maxima near
280 nm. The other 18 amino acids normally found in protein absorb weakly, if at all,
in the UV range.
For this assay you must use one of the Pharmacia/LKB Ultrospec
spectrophotometers on the side bench. The colorimeters at your bench work only in
the visible part of the spectrum; they have no source of UV light. Also, you must use
the special UV-transparent cuvettes because the ordinary plastic cuvettes you use
with your colorimeter are opaque to UV light. The UV-tranparent cuvettes are
marked with small dots of red ink at the top. NOTE: You could use the UVtransparent cuvettes with your colorimeter at visible wavelengths, but you cannot
use the ordinary cuvettes with the LKB instruments in the UV.
There are rather lengthy printed instructions taped to the top of the LKB instruments,
but your instructor may be able to give you a brief demonstration. The wavelength is
set to 280 nm and the instrument is blanked using the "SET REF" button on a
cuvette containing di H2O. (You need to blank the instrument only once.)
You will probably want to use the P 1000 to transfer 1 ml from each standard tube to
the cuvette. Again, work upwards, from lower protein concentration toward higher
protein concentration so that you will not need to rinse the cuvette between samples.
As you read each sample, return it to the tube in case you need to re-read a sample
later.
After you have read all the known protein standards, read the unknown and the
dilutions of the unknown.
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III.
Assemble your data for the known standard solutions in a spreadsheet. You will
have separate columns for proteins concentration (mg/ml), Biuret absorbance
values, and UV absorbance values. Simply ignore any absorbance values < zero.
A. General Comparison of the Assay Methods
The most straightforward way to compare the assay methods to each other (in terms
of their sensitivity and response range) is to draw 2 standard curves on a the same
log/log plot (log Absorbance values vs. log protein concentration). EXCEL makes
this a snap because it eliminates to need to actually calculate logarithms. We urge
you to take advantage of the opportunity to learn how to use EXCEL on the
classroom computers.
Using log scales rather than linear scales on the graph spreads out the data points
that would otherwise be scrunched together at the lower end of the scales. Try it and
see! Note that there is no log value for a negative numbers or for zero. You can
simply eliminate them from the graph. (But you must note in your report that you
have done this.) If you have many negative absorbance values it probably indicates
that the sample concentration is below the detection limit of the method or that there
was a problem with your blank. Also, dirty cuvettes, improper cuvette filling, and
inserting cuvettes in the wrong orientation can cause this problem.
The log/log plot should be labeled Fig. 1. Have your instructor check the plot. They
will tell you whether you should use your own log/log plot for the discussion, or
whether you should refer to the log/log plot provided on the web site.
B. Determining Concentration of the Unknown
Although the log/log plot can be enlightening with respect to the relative merits of the
assay methods (you can immediately see the sensitivity and dynamic response
ranges) the log scales tend to limit the precision with which you can determine the
unknown concentration. To determine the unknown concentration more precisely
you need a linear/linear plot of Absorbance vs.standard concentration that includes
on the dynamic response range of the assay where Beers Law applies.
Plot linear/linear standard curves for the Biuret and UV assays sepatately as Fig. 2
and Fig. 3. Do not simply "connect the dots" in your graphs, but subjectively draw a
best-fit straight line to the points.
When drawing the line you may ignore any points that are obviously erroneous, but
you must explicitly state that you have done so.
When you have the standard curves properly plotted, perform the determinations of
the unknown samples.
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Remember that your determined values are valid on when the absorbance value of
an unknown (or its dilution) lies within the linear response range of the assay, i.e.
that portion of the standard curve that follows Beer's law. You might need to use the
absorbance value of the undiluted unknown, or the absorbance of one of the
dilutions, depending on the assay and on the protein concentration. Expect to
operate to some extent on a trial and error basis.
Summarize the final results in a table in your notebook of the form:
3.14
Dilution used
BIURET ASSAY
Absorbance
Concentration
(mg/ml)
Corrected
Concentration
Dilution used
UV ASSAY
Absorbance
Concentration
(mg/ml)
Corrected
Concentration
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IV.
Which assay method shows the broadest response range and which assay method
is most sensitive? Use the log/log plot (Fig. 1) to evaluate this.
How might the results be affected if a different protein (other than BSA) were used to
create the standard curve?
Evaluate and discuss the pros and cons of each assay method. Do not confine
yourselves to the features described in section I.A.
Did the two assays give similar results for the protein concentration of the
unknowns? If not, which assay do you think is most reliable, and why? The instructor
can provide the actual value of the unknown.
References on Reserve
Gornall, A.G., Bardawill, C. J. and David, M. M. (1949)
Determination of Serum Proteins by means of the Biuret Reaction.
J. Biol. Chem. 177; 751.
Layne, Ennis
Spectrophotometric and Turbidimetric Methods for Measuring Proteins.
Methods in Enzymology, vol. 3, p. 447 (1957)
Peterson, Gary L.
Determination of Total Protein.
Methods in Enzymology, vol. 91, p. 95 (1983)
3.15