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Lab2 BCA Assay Handout
Lab2 BCA Assay Handout
Lab2 BCA Assay Handout
Lab 2
Spring 2011
reduction assays are powerful, linear over a wider range of protein concentrations and are far less
sensitive to the identities of amino acids in the sample, but take longer to do and require more
uncommon chemicals. The bicinchoninic acid (BCA) assay is generally considered to be the
easiest of the copper reduction assays, and is the method we will use in todays experiment.
Regardless of the method used, colorimetric assays require a calibration process prior to
determination of an unknown protein concentration. This process requires the construction of a
standard curve. In this process, the assay is run on several known concentrations of a standard
protein, often bovine serum albumin (BSA) or bovine gamma globulin (BGG), and the
absorbance values for the known concentrations are measured. Ideally, plotting absorbance vs.
concentration will give a linear plot. The assay is then conducted on a protein sample of
unknown concentration, the absorbance is measured, and, assuming the absorbance value is
somewhere within the linear range of the standard curve, the value for concentration that would
correspond to that absorbance is determined. Assuming minimal systematic error, the better
(more linear) the standard curve, the more accurate the concentration determination.
The BCA assay (Figure 1) relies on the temperature-dependent reduction of Cu2+ ions by
peptide bonds in the protein in an alkaline environment. The side chains of the amino acids
cysteine, tyrosine, and tryptophan also participate in the reduction of Cu2+ under alkaline
conditions. The organic dye bicinchoninic acid is then added, which selectively chelates Cu+
ions, leading to the formation of a metal complex with a strong purple color. This Cu+-BCA
complex absorbs light at 562 nm and can be measured with a UV-Vis spectrophotometer.
In this lab, you will test the operating protein concentration range of the BCA assay by
generating a standard curve using known concentrations of BSA. You will assess your data for
its linearity, perform a linear regression, and use your trendline to determine the concentration of
two unknown protein samples.
It is important to note that the color formation in the BCA assay depends on more than
simply the number of peptide bonds present in the sample. It is also affected by the structure of
the protein and the presence of the amino acids cysteine, cystine, tryptophan, and tyrosine. If the
unknown protein sample has similar characteristics to the protein measured in the standard curve,
the concentration of the unknown can be determined with a high degree of accuracy. However, if
the unknown protein sample has many different characteristics to the protein measured in the
standard curve (in size, amino acid composition, or structure), the error in the concentration
determined may be significant. In this lab, the unknown protein sample is actually a sample of
BSA, so you will be comparing the same protein as in your standard curve, which is the ideal
way to determine protein concentrations.
It is also important to be consistent in your experimental technique. Treat all samples
equally. Pipetting should be performed as uniformly and reproducibly as possible. Chemical
reactions generally proceed faster at higher temperatures. It will be particularly important for all
samples to be incubated at elevated temperatures (37 C) for as close to the same time interval as
is practical.
Step 1:
Protein + Cu2+
OH -
Protein + Cu+
COOStep 2:
N
Cu+ + 2 BCA
Cu+
COO-
-OOC
-OOC
Experimental Procedure
Work in groups according to your instructor.
A. Preparation of Working Reagent
1. You will need to make at least 11 mL of working reagent per person in the group.
2. BCA Reagent A contains sodium carbonate, sodium bicarbonate, bicinchoninic acid, and
sodium tartrate in 0.1 M sodium hydroxide. BCA Reagent B contains 4% copper (II)
sulfate. Prepare the working reagent by mixing 50 parts of BCA Reagent A with 1 part
BCA Reagent B in a 50 mL disposable centrifuge tube. Be extremely careful not to
cross-contaminate the Reagent A and Reagent B stocks!
3. Mix well by inverting the capped tube several times. The initial solution will be turbid,
but should make a clear green solution after mixing.
B. Preparation of Standards
1. Your group will run three trials of everything, so you will have to prepare three complete
sets of the above series. Therefore, each member in a group should construct their own
series of standards.
2. In two separately labeled and clean microcentrifuge tubes, obtain a sufficient amount of
the 5.00 mg/mL bovine serum albumin (BSA) stock and a sufficient amount of the 1.00
mg/mL BSA stock solutions.
3. Make nine 200 L standard solutions covering a range of BSA concentrations of 0.00
2.00 mg/mL. Have the following table reproduced and completed in your lab notebook
prior to lab (see Pre-Lab Question 3).
Tube Number
Water Added
(L)
Final [BSA]
(g/mL)
1.00 mg/mL
S0
S1
S2
S3
S4
S5
S6
S7
S8
0
100
200
400
600
800
1000
1500
2000
Your initials8
50
S8
1000
Your initialsU1
50
BSA Unknown
1000
C. Determination of Standard Curve
1. Incubate all samples in a 37C water bath for 30 minutes.
2. While your samples are incubating, initialize and calibrate the Avantes AvaFast Fiber
Optic Diode Array Spectrophotometer (refer to the separate Avantes Spectrophotometer
instructions).
3. When your samples are done incubating, remove them from the water bath, and let them
cool to room temperature.
a. If you do not immediately measure the absorbance, store your incubated samples on
ice.
4. Use the same five cuvets to make all your measurements.
a. Start with your blank sample and progress through the standards from least to most
concentrated. Measure the unknowns last.
b. Pipet 1 mL into the cuvet and measure the absorbance spectrum from 200-700 nm.
Assess the quality of the spectrum (baseline flatness, noise level, peak shape) and
make necessary notes in your observations.
c. Record the absorbance of your samples at 562 nm.
d. After each measurement, return the contents of the cuvet to the sample tube, rinse the
cuvet at least 3 times with DI water, and vigorously shake dry to minimize dilution of
your next sample.
D. Plotting Your Calibration Curve and Determining the BSA Unknown Concentration
(Refer to the appropriate sections in the ECA text for guidelines)
1. For the three sets of standard curve data your group has collected (Trials 1-3), plot A562
vs. BSA concentration (g/mL) in a spreadsheet (one plot per trial).
2. For your individual standard curve, have your spreadsheet plot a trendline to your data
and determine the following using the LINEST function:
Slope and y-intercept of the best linear fit to the data
Equation of the resulting linear fit
R2 value for the resulting linear fit
Error in the slope and y-intercept
Plotting a trendline is done by making the plot and then selecting the data series in the
chart. From the Chart menu, select Add Trendline. In the resulting dialog box, make
sure linear is highlighted. Click the Options tab and click the checkboxes for Display
equation on chart and Display R-squared value on chart. Hit OK. Do not force the
trendline through the origin.
R2 as well as the standard deviation in the calculated slope are measures of how well the
data fits a linear trendline. An R2 value of 1 is perfect fit. As R2 decreases, it indicates the
data fits a linear trendline less well. Use the calculated R2 and errors in the slope to
determine whether omitting suspect points or sets of points significantly improves the
quality of the calibration curve.
Consult with your TA and/or the instructor if you are having difficulty determining
suspect data points. As a rule of thumb, a more accurate trendline gives a y-intercept
close to 0.
3. Using a combination of Grubbs Test and your observations, determine which points to
keep for your final group calibration curve.
a. Take the average of the remaining group A562 values for each BSA concentration
selected and plot this data.
b. Use LINEST to determine the equation for the linear regression and the associated
errors.
4. Use this final calibration trendline to determine the protein concentration of your
unknowns. Report these values with appropriate significant figures, error, and units.
In the discussion section of your notebook, be sure to include:
1. A chemical description of the processes underlying the assay, why you performed the
assay, and what you were able to determine from it.
2. An explanation for your data analysis, including why you used the LINEST function,
why you decided to discard (or not discard) some data points from your curve, and the
precision and accuracy of your data. Generally speaking, how confident are you in the
accuracy of your concentrations of your unknowns?
3. An explanation for any unexpected results and suggestions for how you might improve
the experiment and/or your results if you were to repeat it.
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