Lab #1: Protein Quantitation and Dilutions

LAB #1: PROTEIN QUANTITATION AND DILUTIONS

I. Introduction

In this lab, we will be quantitating the amount of total protein in various samples, using two
methods: the Bradford dye-binding assay and UV absorbance at 280 nm. Both of these assays
use a spectrophotometer to measure the amount of light absorbed by a sample, which can then be
converted to concentration.

Spectrophotometry in the Biochemistry Lab

Many biochemical assays and clinical tests measure the abundance of a particular compound
within a complex sample, for example glucose levels in blood serum. Many biologically
relevant compounds absorb light at particular wavelengths or can be treated with reagents that
react specifically with the compound to produce a colored product. This color formation can be
used to quantify the amount of compound present, using a machine called a spectrophotometer.

A spectrophotometer is an essential part of the biochemistry laboratory because it measures

absorbance of light at a given wavelength. Using Beer’s law, we can convert absorbance into
concentration. If we know this wavelength and a parameter called the extinction coefficient (),
we can accurately determine the concentration of our compound.

Beer’s Law: A = bc where A (or Abs) = absorbance at a given wavelength (unitless)
 = extinction coefficient ((mg/mL)-1 cm-1)
b = path length of cuvette (cm)
c = concentration (mg/mL)

Since  and b are constants for a given molecule and spectrophotometer, absorbance is directly
proportional to concentration: Abs ∝ conc.

However, often we do not know the extinction coefficient for our compound. In this case, we
can use a standard curve to determine the relationship between Absorbance and Concentration.

A standard curve is constructed by carrying out the procedure using diluted solutions of the
compound of interest with known concentrations. The absorbance of our known standards is
measured and a graph is generated of Absorbance as a function of Concentration:

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 Lab #1: Protein Quantitation and Dilutions

We can then measure the absorbance of our samples of unknown concentration and place them
onto the graph. We can estimate the concentration by using a ruler and drawing a line down the
x-axis.

Or using curve-fitting equations in any graphing program (such as LoggerPro or Excel), we can
precisely calculate the concentration of our compound of interest in each sample.

Important considerations:
 Your sample must be within the range of concentration of the standards.
o For the above curve, if the absorbance of your sample was above 1.0, you would
need to include another standard of a concentration > 1 mg/mL. Or you would
need to dilute back your sample to obtain an absorbance within the standard
curve.
o If the absorbance of your sample was below 0.2, you would need to include
another standard of a concentration < 0.2 mg/mL. Alternatively, this may be
outside of the limit of detection of this assay and you may need to find another,
more sensitive assay.
 The absorbance of your sample should preferably lie within the range in which the graph
of the standard curve is linear. Generally speaking, this means the absorbance of your
sample should be less than 1.0. If it is greater than 1.0, make an appropriate dilution and
repeat the procedure. After determining the absorbance and concentration of the diluted
sample, multiply that concentration by the dilution factor you used to calculate the
concentration of the analyte in the original sample.
 Your standards should be prepared in the same buffer as your unknown sample in
order to eliminate aberrantly high or low readings due to interference of detergents, salts,
chelators, or other buffer components.

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 Lab #1: Protein Quantitation and Dilutions

Bradford (Bio-Rad) assay

One common way to measure the amount of protein in a sample is to use one of several
colorimetric, dye-binding assays. These dye-binding assays rely on the fact that certain
compounds bind to proteins non-specifically but quantitatively and change color upon binding.

 Non-specifically means that the reagent binds to all (or most) proteins equivalently.
(Note that there is some influence of certain amino acids, notably Arg, Lys, and His for
Coomassie-based assays or Trp, Tyr, and Cys for copper-based assays, so it is possible
for certain proteins to have unusually strong or weak reactions with the dyes.)
 Quantitatively means that the more protein, the more binding and the more color
change, and that signal is linearly proportional to the amount of protein.
 The color change allows scientists to monitor only the portion of the dye reagent that is
associated with proteins and ignore the excess dye that is not bound to protein. It also
makes possible an easy, quick visual test for the presence of protein.

There are several variations on the dye-binding assays, using different dye reagents and
optimized for different buffer conditions, wavelengths of detection, or protein concentration
ranges. The most common reagents are: Coomassie blue dye (Bradford and Bio-Rad assays)
or copper ion solutions (Lowry and BCA assays).

The assays using Coomassie blue dye are particularly sensitive to detergents in the buffer, which
compete for binding to the proteins and strongly decrease the color change. The copper-based
assays are inhibited by metal chelators, while reducing agents and free thiols cause a strong
background signal. These are particular issues since cells are often lysed in solutions containing
detergents, chelators, and reducing agents. Other buffer components can also affect the assays
(see Table 1), so before performing any of these assays, a thoughtful scientist will check the
known ingredients in a sample for compatibility with the particular assay and select a
different method if necessary.

Table 1: Maximum compatible concentrations of a few common buffer reagents

Compound Bradford Assay Lowry Assay BCA Assay
EDTA (chelator) 100 mM 1 mM 10 mM
Glycerol 10 % 10 % 10 %
Sucrose 10 % 7.5 % 40 %
-mercaptoethanol 1M 1 mM 0.01%
Sodium chloride 5M 1M 1M
Sodium phosphate 100 mM 100 mM 100 mM
Sodium deoxycholate 0.05 % n/a 5%
SDS (detergent) 0.125 % 1% 5%
Triton X-100 (detergent) 0.125 % 0.031 % 5%
Tris-HCl 2M 10 mM 250 mM

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 Lab #1: Protein Quantitation and Dilutions

In this lab, we will be using the Bio-Rad version of the Bradford assay, using Coomassie blue
binding to proteins. By itself, the Coomassie dye is a reddish-brown color (absorbance
maximum at 465 nm), but upon binding to proteins, a spectral shift occurs producing a blue
color (absorbance maximum at 610 nm). We can visualize this directly by eye but will use a
spectrophotometer to quantitatively measure the amount of color change. The optimal
wavelength to measure the difference is 595 nm.

The Coomassie dye is dissolved in an acidic solution, so take care when handling. Also, the dye
will form small aggregates over a period of minutes, so mix the solution by inversion before
removing it from the stock bottle. Also, mix your group’s aliquot well before adding it to each
tube of protein solution.

First, you will prepare a set of standards of protein samples of known concentration. The protein
we will use is bovine serum albumin (BSA), a common protein that is easily purified. (Another
frequently used standard is bovine gamma globulin (BGG or IgG).) You will prepare dilutions
of BSA from a concentrated stock in the same buffer (PBS) as your samples. Then, you will add
the Bio-Rad reagent (a proprietary formulation of acidic Coomassie dye), mix well, and measure
absorbance at 595 nm (A595) in the spectrophotometer.

UV Absorbance at 280 nm

We will also perform an independent measure of protein concentration using the intrinsic UV
absorption of two amino acids.

Trp and Tyr,

two of the
aromatic
amino
acids,
have maximal

absorbance at 280 nm. Thus, in a solution which has few other compounds that absorb at 280
nm, we can measure A280 and convert to concentration.

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 Lab #1: Protein Quantitation and Dilutions

To do this, we will need to use the extinction coefficient, . This can be empirically measured or
calculated using the number of Trp and Tyr residues a protein has:
 (M-1 cm-1) = (# Trp x 5500) + (# Tyr x 1490)
If you do not know the sequence of the protein, however, we can use a rough estimate of
 ~ 1 (mg/mL)-1 cm-1. Most proteins have  values between 0.4 – 2.4 (mg/mL)-1 cm-1, so this is a
reasonable estimate for some purposes. However, since different proteins can vary considerably
in their content of Trp and Tyr, using an estimate of  ~ 1 is not sufficiently accurate for highly
quantitative applications.

A common use of A280 measurements is during protein purifications. Chromatography machines

called FPLCs have a UV detector that measures the A280 of the solution as it exits the column.
This allows scientists to observe the amount of protein in each fraction as it is collected. The
A280 trace below shows that most of the proteins in this purification did not bind and flowed
through the column. A wash step removed non-specifically bound proteins, and in the last step,
the protein of interest was eluted when the salt concentration was increased.

In this lab, you will use A280

measurements to find the concentration of your protein samples.

When to use which assay?

There are advantages and disadvantages to each protein quantitation assay. Dye-binding assays
can be very sensitive. Due to non-specific binding of the dye (Coomassie or Cu reagents), all
proteins are detected. However, certain chemicals can interfere with the assay. A280
measurements are simple, quick, and can be used with a wider range of buffers. However, they
are less sensitive. They also rely on detecting Trp and Tyr, so if a protein lacks these amino
acids or if the amino acid sequence is not known, the quantitation may be less accurate.

Advantages Disadvantages
Dye-binding assays  Rapid  Chemicals can interfere (SDS,
 Detects all proteins EDTA)
 Very sensitive (~1 g/mL)
A280 measurement  Very rapid  Detects only Trp and Tyr
 Simple  Not as sensitive (~0.1 mg/mL)
 Less interference from chemicals
Pipetting, Dilutions, and Mixing

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 Lab #1: Protein Quantitation and Dilutions

In order to quantify biomolecules, you need to make standard (calibration) curves and/or
measure multiple dilutions of the sample. Thus, your quantitation will only be as good as your
pipetting and solution making skills! Fortunately, we will practice this a lot this semester.

See the Pipetting and Dilutions hand-outs for more information.

When making any solution, you must always mix well! This includes after making a solution,
after making a dilution, and also after thawing out a frozen sample. When in doubt, mix!

Ways to mix solutions include:

 Using a magnetic stir bar and spinning on a stir plate.
 Capping the sample and shaking or inverting the bottle or tube.
 Pouring the solution back and forth between containers. (Not ideal, but can do in a
pinch.)
 Vortexing the solution.
 Pipetting up and down. (This is useful for gentle mixing.)
 Flicking the tube. (This is useful for gentle mixing.)

Unknowns

In this lab, you will be using the Bradford assay and A280 values to determine the concentration
of unknown protein solutions.

 Pure proteins (Unknowns A and B): often after a protein purification, we need to
quantitate the amount of our purified protein for downstream applications or experiments,
for example:
 To prepare purified Taq DNA polymerase as a PCR reagent
 Kinetic analysis of a new enzyme that synthesizes a drug

 Complex protein solutions (E. coli lysate, body fluids or tissues (blood, urine) or dietary
products (milk, protein drinks): we often need to know the amount of protein for
diagnostic purposes or before starting a procedure like SDS-PAGE.

Sample Organization and Management

In this lab, you will have many samples and dilutions. It is important to communicate in your
group and keep track of samples to avoid mix-ups! Helpful hints:
 Label tubes.
 Always keep samples in an order. Do not just put them randomly into a rack.
 When adding multiple components to a tube, set up tubes in the back row of a rack. After
adding a reagent to a tube, move it forward in the rack.

Workflow of the Lab

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 Lab #1: Protein Quantitation and Dilutions

A. Bio-Rad Assay (Bradford Assay)

B: UV Absorbance at 280 nm

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 Lab #1: Protein Quantitation and Dilutions

Note that you will not need your standards for this part of the experiment! You also will not
need the Unknown mixed with the dye. Instead, you will simply use the original Unknown
sample, or (if needed) dilutions of the Unknown.

Examples of good, bad, and ugly spectrophotometer results:

Out of linear range of

spectrophotometer
Poorly defined peak at
280 nm, suggests
interference from other
Baseline not molecules besides protein
Great data! at zero

 Example of good data.  Repeat this measurement,  Not much you can do here.
diluting down sample.  Data is useable, but not high-
 Also may need to reblank to quality. Note this in results.
get absorbance at the baseline
back down to zero.

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 Lab #1: Protein Quantitation and Dilutions

C. Using the spectrophotometers

In biochemistry, we typically use 1 mL cuvettes which have indentations to reduce their volume
from the larger 5 mL cuvettes. The clear, 1 cm width side of the cuvettes must be oriented in
the direction of the light path of the spectrophotometer.

Arrow.
Clear side

 For the cuvettes with a ridged surface, the clear side should face the light source (arrow).
 For the cuvettes which do not have ridges , the arrow should face the light source (arrow).

 Correct!  Wrong way.  Correct!.

 Clear side facing light  Ridged side facing light,  Arrow on cuvette is lined up
(arrow). will interfere with reading. with the arrow on spec.

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 Lab #1: Protein Quantitation and Dilutions

II. Protocol

Obtain:
 5 mL Bio-Rad dye concentrate. Gently invert the bottle 10 times before pipetting the dye
solution out to mix and dissolve any dye aggregates. Pour only the amount needed into
a beaker or tube and take to your bench.
 1 mL BSA (Bovine Serum Albumen) stock solution (1.0 mg/mL)
 ~25 mL PBS (Phosphate Buffered Saline, a common lab buffer)
 Pure protein solutions: 5 mL of Unknown A and B
 1 mL cuvettes (for the spectrophotometer). We may re-use these. Clean with 0.2% SDS.
 1.5 mL and 2 mL microcentrifuge tubes. Remove extra solution to hazardous waste, then
dispose of in trash. Use the 1.5 mL tubes for most purposes. The 2 mL tubes are only
needed for serial dilutions.

Other items:
 50 L UV-transparent cuvettes: located by the UV-visible spectrophotometers.

In this lab, you will carry out two independent measurements of protein concentration using two
different assays, and then compare the two methods.

A. Bio-Rad Assay (Bradford Assay)

(Reference: Marion M. Bradford (1976). Analytical Biochemistry 72: 248–54.)

1. Prepare 5 standards of known concentrations of BSA ranging from approximately 2 g/mL to

25 g/mL.
 Make two duplicate solutions of each concentration in a final volume of 1 mL. Prepare
these in 1.5 mL (for a) or 2 mL (for b) tubes. Use PBS to dilute the BSA stock solution.
 You may do this in one of two ways:
a) Choose 5 concentrations and use the dilution equation (c1)(v1) = (c2)(v2) to
determine how to prepare these. Note: it is easiest if you choose round
numbers.
b) Prepare a 24 g/mL standard. Then make serial 2-fold dilutions to get 12, 6, 3 and
1.5 g/mL. In this case, be sure to prepare 2 mL of each dilution (except the last) so
that you have enough to use to make the next 2-fold dilution and still have 1 mL
remaining.
 Be sure to mix the dilutions well at each step! (Since these have caps, you can vortex.)

2. Measure A595 values for the standards of known concentration.

 The Bio-Rad dye should be added to the sample to make a 1:5 dilution (1 part dye in 5
parts total). Although we will only transfer 1 mL to the cuvette, today we will use a final
volume of 1.125 mL to leave a little extra for pipetting purposes.
 Add 900 L of each standard to a 1 mL cuvette. Use a cuvette rack to keep samples in
order. You can put the rack on a piece of paper and record the sample information there.
You will have duplicates for each standard concentration.
 For a blank, add 900 L buffer to a tube. (Only one blank is needed.)

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 Add 225 L of the Bio-Rad dye concentrate to each tube, including the blank! Vortex or
pipette up and down several times to mix. (You can also cover with parafilm and invert
the cuvettes.)
 Incubate reactions 5 or more minutes at room temperature. (Note that the color will
continue to slowly develop over time, so all samples should be read at once.)
 Put the blank solution into the spectrophotometer set for 595 nm.
 Set the reference to zero.
 Measure the absorbance of each standard at 595 nm.
 Record this data in your lab notebook and in the tables in the Post Lab.
 Check with the instructor to make sure the data are reproducible between duplicates and
within the expected Abs595 range. If not, your pipetting skills may need to be improved!

3. Prepare and measure A595 values for the samples of unknown concentration.
 After the standards have been measured (above), then measure the samples by mixing
900 L of Unknown A or B with 225 L of the Bio-Rad dye concentrate as above.
 You may need to try multiple different dilutions to obtain absorbance values within
the linear range of your standards! (Refer to Pre-Lab Q4 or above.)
 If so, use PBS to dilute the unknown solutions. (Always use the same diluent for your
standards as your unknowns.) Remember to mix the dilutions!
 To find the appropriate dilution, at first make one 1 mL dilution in a 1.5 mL tube. If the
Abs595 is too low or too high, try another dilution.
 Once you have determined a useful dilution, prepare two duplicate aliquots of that
dilution and measure both aliquots to ensure reproducibility.
 Record this data in your lab notebook and in the tables in the Post Lab. Make sure to
record the dilutions used, if any!

You have obtained all the needed raw data for this method. However, you do not yet know the
concentration of your unknown samples. You are about to determine these in Step 4!

4. Graph your data. (You may need to do this at home.)

 Set up a graph with A595 on the y-axis and concentration on the x-axis. (See below.)
 Plot your standards. For duplicate standards that were similar in absorbance readings,
use the average value. If any standards gave significantly different readings, consider
why this might be. Try graphing both and see which value seems more likely to be
accurate.
 First, estimate the concentration. Make sure to take the dilution factor into account!
 To estimate the concentration, draw your sample data on the graph (see top of pg. 2).
You can insert the graph into a document and then draw on the image in Word by
copying and pasting the graph from Excel. (You may want to use “paste special > pdf”.)
Or you can print out your graph and draw by hand on the printout. Again, use the
average value, unless you obtained significantly different readings.
 Second, calculate the exact concentration of your samples. Make sure to take the
dilution factor into account!
 To calculate the exact concentration, use the graphing software. Use a best fit line (in
Microsoft Excel this is called a “trendline”) to obtain the equation relating the y-axis

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 Lab #1: Protein Quantitation and Dilutions

(A595) to the x-axis (concentration). Include the equation and the R2 value or correlation
coefficient. (A perfect R2 value is 1.)
 Copy and paste your new graph with the best fit line, equation, and R2 value into your
Word document, or print out your new graph.

Enter the estimated and calculated concentrations in your lab notebook and in the tables in the
Post-Lab. Turn in both of your graphs and calculations with your Post-Lab this week.

Bonus: Making good graphs.

 Use professional spreadsheet program, like Excel, LoggerPro, or Google Sheets.
 Making a good graph starts with having a good, well-organized data table. Make sure to
label the columns in your table with headings containing units.
 Make sure to choose “Scatter plot”. Do not use “Line chart” (since in most programs, it
does not use your data points for the x-axis.)
 Give the graph a descriptive title, as well as axis labels with units. For absorbance
values, include the wavelength.
 If you have more than one line, make sure to include a legend.

Pro Tip: Scaling Assays Up or Down

 For many assays, you can scale up or down the volume depending on your needs.
 For example, we will use the Bradford Assay later with 800 L of sample.
 How will we calculate how much dye to use?
 Must keep the same ratio of components! Here, we know the new sample volume, so can use the dye:sample ratio.

225 μ L dye x μL dye

=
900 μ L sample 800 μL sample

 We will also use a microplate Bradford Assay with a total volume of 125 L.
 How will we know how much sample and dye to use?
 Here, we know the total volume, so can use the dye:total ratio.

225 μL dye x μL dye

=
(900+225) μ L total 125 μL total

 Note that the dye/total ratio above is 225/1125, which is the same as 1/5.
 So all of the above set-ups are using 1 part dye + 4 parts sample = 5 parts total, or a 1:5 dilution!

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 Lab #1: Protein Quantitation and Dilutions

B: UV Absorbance at 280 nm

In the previous assay, we indirectly measured our proteins via the color change of a dye that
bound to the proteins. For this assay, we will be directly measuring the proteins via the Trp and
Tyr amino acids absorption of UV light. Thus, we do not need a standard (calibration) curve.
Instead, we can simply convert the Abs280 to concentration using an equation (Beer’s Law).

1. Prepare the samples of unknown concentration. Use PBS to dilute the samples.
 Refer to Pre-Lab Q5 for the range of useful concentrations for the A280 assay.
 Start with undiluted solutions of the unknowns. You will need 60 L for each
measurement. You may need to make dilutions, based on your initial A280 values.
 You may need to try multiple different dilutions to obtain samples with absorbances in
the linear range of the spectrophotometer.

2. Measure A280 values using the 50 L UV-transparent cuvettes.

 For this part, we must use a spectrophotometer that can detect in the UV wavelengths
(<400 nm), either the spectrophotometer from Dr. Waters’ research lab (on the side
bench) or the diode-array UV-Vis spectrophotometer (in the side room).
 Transfer 60 L of your buffer to a UV-transparent cuvette (kept near the UV-vis
spectrophotometer). This will be your blank.
 Set the spectrophotometer to 280 nm and zero the machine on your blank.
 Rinse the cuvette out and re-use for each sample, rinsing well inbetween.
 Measure the absorbance at 280 nm (A280) for each sample.
 Record this data in your lab notebook and in the tables in the Post-Lab. Make sure to
record the dilutions used, if any!

3. Calculate the concentration of your samples.

 Using Beer’s Law, calculate the concentration of your unknown samples using the
generic extinction coefficient  = 1 mg-1 mL cm-1. Make sure to take dilution factors
into account!
 Compare your concentration values obtained in Part A with those in Part B.
 In the Post-Lab, the identity of the unknown proteins will be revealed and you can use the
specific value from the literature to calculate a more accurate concentration.

Enter the calculated concentrations in your lab notebook and in the table in the Post-Lab.

Safety and Clean Up

The Bio-Rad reagent stains proteins (e.g., fingers and clothes). You may want to wear gloves
and a lab coat.

The Bio-Rad reagent contains phosphoric acid. Dispose of solutions in the hazardous waste
container in the hood.

Clean tubes and cuvettes with 0.2% SDS solution or dilute soap to remove remaining dye.
Rinse well afterwards to remove detergent residue.

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