Select a Scientific peer reviewed paper Data Analysis (100 points total):A) Presentation of Data/Results (20 points total)Include all actual raw data sets from your experiment. Examples include charts, graphs, gel pictures, blots, and/or fluorescence images. Properly label all data sets. (20 points)Make sure to title each piece of data and include proper labels or identification keys. Your 20 points for data sets will be graded as follows:16-20 pts. – All results are presented, clearly labeled, and separated into sub-areas with detailed and accurate data. It may include charts, graphs and/or images.11-15 pts. – All results are presented, clearly labeled and separated with few data inaccuracies. It may include charts, graphs and/or images.6-10 pts. – Most results are presented, haphazardly labeled with several inaccuracies.1-5 pts. – Some results are presented, unlabeled with a poor quality appearance.0 pts. – The results are missing.B) Explanation of Data/Results (30 points total)Text description of obtained results. (15 points total)Explain how you generated your standard curve. (5 points)Explain how your calculated your dilution factors. (5 points)Explain how you calculated your sample protein concentration. (5 points)Description of obtained results vs. expected results. (3 points)Explanation of obtained results. Why did you get these results? Use scientific reasons to explain all of your data. (10 points)How would you improve your protocol if you repeated your experiment? (2 points)C) Discussion (40 points total)What was the goal of this experiment? (5 points)Write a brief summary supporting or refuting your hypothesis from your protocol. (10 points)What controls were used in the experiment? (2 points) Did the controls work? Explain. (3 points)Write a brief summary about what you can conclude from the data/results. (15 points)Briefly describe your next experiment and the questions that it will answer about your protein sample. (5 points) Your 15 points for your discussion summary will be graded as follows:14-15 pts. – All results are clearly interpreted, hypotheses are made and evaluated for the obtained results and their deviation from the “expected” results, if necessary; all experimental questions are answered thoughtfully and completely. Overall, the writing is concise, complete and of outstanding quality grammatically. There is a good synthesis of the experiment and the results, particularly in the scope of a larger context.11-13 pts. – All results are clearly interpreted, hypotheses are made and evaluated for the obtained results and their deviation from the “expected” results, if necessary; all experimental questions are answered thoughtfully and completely. Overall, the writing is concise, complete and of high quality grammatically. The synthesis of the experiment and the results are limited to the context of the experiment.8-10 pts. – Most results are addressed, though hypotheses for unexpected results are weak and not fully explored; experimental questions are not answered with well-formulated or complete answers. The writing is not concise or complete and is of good quality.6-9 pts. – Most results are addressed; hypotheses for unexpected results are missing; experimental questions are not adequately answered. The writing is of fair quality. The synthesis of the experiment is limited to the experiment.3-5 pts. – Some results are addresses; hypotheses for unexpected results are missing; experimental questions are not answered. The writing is of poor quality. Synthesizing thoughts about the experiment and its context are absent.1-2 pts. – Results are acknowledged; hypotheses are missing; experimental questions are unanswered. The writing is of poor quality and synthesizing thoughts are missing.0 pts. – The results are missing.D) In text citations (5 points total)E) References page (5 points total)MCB 253
Protein Quantitation
© Elizabeth A. Good
University of Illinois at Urbana-Champaign
S
What is the concentration of our
protein sample?
S What do you get from protein purification?
S Pure protein sample
S What do you do with your pure protein sample?
S Run experiments
S Why quantify before running experiments?
S What does quantitation tell us about the protein sample?
S How much protein you have/concentration
2
What properties of proteins are
utilized for quantitation?
S Different molecules absorb different wavelengths.
S UV Spectrophotometry → Proteins absorb UV
S
280nm (Aromatic), 200nm(peptide)
S Bradford Assay
S
S
Coomassie blue dye binds to Arg, Aromatic amino acids → Turns blue
Dye absorbs at 595nm
3
Spectrophotometry
S The absorbed light is related to concentration.
S Beer’s Law → = × ∁ ×
S (Absorbance = Molar Absorptivity X Concentration X path length)
S Path length = 1cm ; = × ∁
S A∝C
4
Spectrophotometer
S 7 samples in cuvettes
S 1 blank + 6 protein samples
S
Standard protein (BSA)
S
Your protein sample ([unknown])
S Optical window on cuvette
S Set single wavelength at 595nm
S Optical beam
5
Bradford Assay
S
Why dilute the protein samples?
S
Importance of Bradford Reagent to protein sample ratio (50:1)
S
Total sample volume depends on cuvette size = 1.2ml = 1200ul
S
Volume of diluted protein sample (protein+buffer) ~ 24ul
S
Volume of Bradford Reagent up to 1.2ml
S
Each sample will contain 1.2ml Bradford Reagent plus 24ul protein dilution.
6
Calculations of Dilutions
S Concentration, (
)
=
ℎ ( )
( )
S 1 × 1 = 2 × 2
S =
S =
1
=
=
1
2
2
1
; C2 = C1 ×
; 1 = 2 ×
S Use any ONE of these! They are all the same.
7
Experimental Design
S Prepare dilutions:
S 6 dilutions for standard protein (Bovine Serum Albumin protein
(BSA)- known concentration of 2mg/ml)
S 6 dilutions for your protein (unknown concentration- what we are
trying to determine)
S Use varied dilutions – why?
S Use a blank – why is it important?
8
Analyzing the DataCreating a Standard Curve
(Absorbance vs. Concentration)
From the spectrophotometer, what
does each concentration give us?
S
Absorbance
S
Absorbance
S
Plot Absorbance vs. Concentration for
BSA standard protein→ Standard
curve
S
Find absorbance of protein samples
(unknown concentration)
S
Use standard curve to find out
concentration of your protein
Line of
best fit
Concentration
9
Good Lab Practices
S Thawing reagents – why?
S Mixing the reagents well – why?
S Avoiding Bubbles – why?
S Accurate Pipetting – why?
S Temperatures and times – importance?
10
TA Help Sessions
S Day: Friday
S Time: 10am-1pm or 2-5pm
S Location: Online in Zoom
(Link posted on our course Moodle page)
11
Upcoming Week 3 Activities
Week 3: February 8-12, 2021:
S Bradford Assay Experiment
S Data Analysis Group Work
12
Upcoming Assignment Deadlines
Due on Friday, February 5, 2021:
S Protein Purification Protocol
S Five Protein Purification Peer-reviewed Research Papers
S Protein Purification References Page
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™
Quick Start Bradford
Protein Assay
Instruction Manual
For technical service call your local Bio-Rad office, or in the US,
1-800-4BIORAD (1-800-424-6723)
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Table of Contents
Section 1
Introduction
1
1.1 Principle
1
1.2 Selecting a Protein Standard
5
1.3 Product Description
Section 2
Instructions
9
11
2.1 Standard Assay Protocol
11
2.2 Microassay Protocol
14
Section 3
Data Analysis
18
Section 4
FAQs and Troubleshooting
22
Section 5
Ordering Information
26
Section 6
References
28
Section 7
Appendix
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Section 1
Introduction
The Quick Start Bradford protein assay is a
simple and accurate procedure for determining
the concentration of protein in solution. It provides
ready-to-use convenience by supplying the dye
reagent at 1x concentration and two protein assay
standards at seven prediluted concentrations. The
prediluted standards are conveniently packaged in
2 ml screwcap vials, eliminating wasteful and
sharp ampoules, and ensuring protein stability
over the shelf life of the product.
1.1 Principle
The Bradford assay is a protein determination
method that involves the binding of Coomassie
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Brilliant Blue G-250 dye to proteins (Bradford
1976). The dye exists in three forms: cationic
(red), neutral (green), and anionic (blue) (Compton
and Jones 1985). Under acidic conditions, the
dye is predominantly in the doubly protonated red
cationic form (Amax = 470 nm). However, when the
dye binds to protein, it is converted to a stable
unprotonated blue form (Amax = 595 nm) (Reisner
et al. 1975, Fazekes de St. Groth et al. 1963,
Sedmack and Grossberg 1977). It is this blue
protein-dye form that is detected at 595 nm in the
assay using a spectrophotometer or microplate
reader.
+
H
Cation
«
470 nm (red)
+
H
Neutral form
« Anion
650 nm (green) 595 nm (blue)
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Work with synthetic polyamino acids indicates
that Coomassie Brilliant Blue G-250 dye binds
primarily to basic (especially arginine) and
aromatic amino acid residues (Compton and
Jones 1985). Spector (1978) found that the
extinction coefficient of a dye-albumin complex
solution was constant over a 10-fold concentration range. Thus, Beer’s law may be applied for
accurate quantitation of protein by selecting an
appropriate ratio of dye volume to sample
concentration.
Certain chemical-protein and chemical-dye
interactions interfere with the assay. Interference
from non-protein compounds is due to their ability
to shift the equilibrium levels of the dye among the
three colored species. Known sources of interference,
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such as some detergents, flavonoids, and basic
protein buffers, stabilize the green neutral dye
species by direct binding or by shifting the pH
(Compton and Jones 1985, Fanger 1987).
Nevertheless, many chemical reagents do not
directly affect the development of dye color when
used in the standard protocol and the more
common reagents are listed in Table 1. The
microassay is compatible with lower concentrations of reagents 1/25 than listed in Table 1 due to
the larger sample volume-to-dye ratio. Since every
protein-chemical reagent combination has not
been assayed, it is possible that some of the listed reagents interfere in combination with certain
proteins. However, with respect to proteins such
as bovine serum albumin (BSA) and bovine
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gamma–globulin, the listed reagents show little or
no interference.
1.2 Selecting a Protein Standard
In any protein assay, the ideal protein to use as a
standard is a purified preparation of the protein
being assayed. In the absence of such an
absolute reference protein, another protein must
be selected as a relative standard. The best
relative standard to use is one that gives a color
yield similar to that of the protein being assayed.
Selecting such a protein standard is generally
done empirically. Alternatively, if only relative
protein values are desired, any purified protein
may be selected as a standard. The two most
common protein standards used for protein
assays are BSA and gamma-globulin.
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With the Quick Start Bradford protein assay, dye
color development is significantly greater with
BSA than with most other proteins, including
gamma–globulin. Therefore, the BSA standard
would be an appropriate standard if the sample
contains primarily albumin, or if the protein being
assayed gives similar response to the dye. For a
color response that is typical of many proteins,
the gamma–globulin standard is appropriate.
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Table 1. Reagents compatible with the
Quick Start Bradford protein assay when
using the standard procedure.*
Acetone, 10%
Acetonitrile, 10%
Ammonium sulfate, 1 M
Ampholytes, 3–10, 0.5%
ASB-14, 0.025%
Ascorbic acid, 50 mM
Bis-Tris, pH 6.5, 0.2 M
b-mercaptoethanol, 1 M
Calcium chloride, 40 mM
CHAPS, 10%
CHAPSO, 10%
Deoxycholic acid, 0.2%
DMSO, 5%
Dithioerythritol (DTE), 10 mM
Dithiothreitol (DTT), 10 mM
Eagle’s MEM
Earle’s salt solution
EDTA, 0.2 M
EGTA, 0.2 M
Ethanol, 10%
Glucose, 20%
Glycerol, 5%
Glycine, 0.1 M
Guanidine-HCl, 2 M
Hank’s salt solution
HCl, 0.1 M
HEPES, 0.1 M
Imidazole, 0.2 M
Magnesium chloride, 1 M
MES, 0.1 M
Methanol, 10%
Modified Dulbecco’s PBS
MOPS, 0.1 M
NAD, 2 mM
Nonidet P-40, 0.25%
Octyl b-glucoside, 0.5%
Octyl b-thioglucopyranoside, 1%
PBS
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Phenol Red, 0.5 mg/ml
PIPES, 0.2 M
PMSF, 2 mM
Potassium chloride, 2 M
Potassium phosphate, 0.5 M
SB 3–10, 0.1%
SDS, 0.025%
Sodium acetate, pH 4.8, 0.2 M
Sodium azide, 0.5%
Sodium bicarbonate, 0.2 M
Sodium carbonate, 0.1 M
Sodium chloride, 2.5 M
Sodium citrate, pH 4.8 or
6.4, 0.2 M
Sodium hydroxide, 0.1 M
Sodium phosphate, 0.5 M
Sucrose, 10%
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TBP, 5 mM
TBS (25 mM Tris, 0.15 M NaCl,
pH 7.6), 0.5x
TCEP, 20 mM
Thio-urea, 1 M
Tricine, pH 8, 50 mM
Triethanolamine, pH 7.8, 50 mM
Tris, 1 M
Tris-glycine (25 mM Tris, 192 mM
glycine)
Tris-glycine-SDS, (25 mM Tris,
192 mM glycine, 0.1% SDS),
0.5x
Triton X-100, 0.05%
Tween 20, 0.01%
Urea, 4 M
Coomassie is a trademark of Imperial Chemical Industries. Triton is a trademark
of Union Carbide Corp. Tween is a trademark of ICI Americas, Inc.
*The concentration limits for compatibility with the microassay are 1/25 of the
values in Table 1.
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1.3 Product Description
All kit components have a 1 year shelf life at 4°C.
Standards are provided in a 0.9% NaCl, 0.05%
NaN3 solution.
1x Dye Reagent: 1 L of dye solution containing
methanol and phosphoric acid. One bottle of dye
reagent is sufficient for 200 assays using the
standard 5 ml procedure or 4,000 assays using
the microplate procedure.
BSA Standard, 2 mg/ml: Provided in 2 ml
tubes.
Bovine Gamma-Globulin Standard, 2 mg/ml:
Provided in 2 ml tubes.
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Bovine Serum Albumin Standard Set:
Set of 7 concentrations of BSA (2, 1.5, 1, 0.75,
0.5, 0.25, 0.125 mg/ml) in 2 ml tubes.
Bovine Gamma-Globulin Standard Set:
Set of 7 concentrations of gamma–globulin (2,
1.5, 1, 0.75, 0.5, 0.25, 0.125 mg/ml) in 2 ml
tubes.
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Section 2
Instructions
2.1 Standard Protocol
1. The standard protocol can be performed in
three different formats, 5 ml and a 1 ml
cuvette assay, and a 250 µl microplate assay.
The linear range of these assays for BSA is
125–1,000 µg/ml, whereas with gammaglobulin the linear range is 125–1,500 µg/ml.
2.
Remove the 1x dye reagent from 4°C storage
and let it warm to ambient temperature. Invert
the 1x dye reagent a few times before use.
3.
If 2 mg/ml BSA or 2 mg/ml gamma-globulin
standard is used, refer to the tables in the
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appendix as a guide for diluting the protein
standard. (The dilutions in the tables are
enough for performing triplicate measurements of the standards.) For the diluent, use
the same buffer as in the samples (refer to
Troubleshooting section for more information). Protein solutions are normally assayed
in duplicate or triplicate. For convenience,
the BSA or gamma-globulin standard sets
can be used, but blank samples (0 µg/ml)
should be made using water and dye
reagent.
4.
Pipet each standard and unknown sample
solution into separate clean test tubes or
microplate wells (the 1 ml assay may be
performed in disposable cuvettes). Add the
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1x dye reagent to each tube (or cuvette) and
vortex (or invert). For microplates, mix the
samples using a microplate mixer. Alternatively, use a multichannel pipet to dispense
the 1x dye reagent. Depress the plunger
repeatedly to mix the sample and reagent in
the wells. Replace with clean tips and add
reagent to the next set of wells.
5.
Assay
Volume of
Standard and Sample
Volume of
1x Dye Reagent
5 ml
1 ml
Microplate
100 µl
20 µl
5 µl
5 ml
1 ml
250 µl
Incubate at room temperature for at least
5 min. Samples should not be incubated
longer than 1 hr at room temperature.
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Set the spectrophotometer to 595 nm. Zero
the instrument with the blank sample (not
required for microplate readers). Measure the
absorbance of the standards and unknown
samples. Refer to Section 3 for data analysis.
Note: If the spectrophotometer has a
reference and sample holder, the instrument
can be zeroed with two blank samples. If the
effect of buffer on absorbance is required,
zero the instrument with a cuvette filled with
water and dye reagent in the reference
holder.
2.2 Microassay Protocol
1. The microassay protocol can be performed in
two different formats, a 2 ml cuvette assay
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and a 300 µl microplate assay. The linear range
of these assays for BSA is 1.25–10 µg/ml,
whereas with gamma–globulin the linear range
is 1.25–20 µg/ml.
2.
Remove the 1x dye reagent from the 4°C
storage and let it warm to ambient temperature. Invert the 1x dye reagent a few times
before use.
3.
Depending on the type of standard used, refer
to the tables in the appendix as a guide for
diluting the protein standard. For the
diluent, use the same buffer as in the samples.
Protein solutions are normally assayed in duplicate or triplicate. The dilutions in the tables
provide enough volume to run triplicates.
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Pipet each standard and unknown sample
solution into separate clean test tubes,
disposable cuvettes, or microplate wells. Add
1x dye reagent to each tube or cuvette and
vortex: for microplates, mix the samples
using a microplate mixer. Alternatively, use a
multichannel pipet to dispense the 1x dye
reagent. Depress and release the plunger
repeatedly to mix the sample and reagent in
the wells. Replace with clean tips and add
reagent to the next set of wells.
Assay
Volume of
Standard and Sample
Volume of
1x Dye Reagent
2 ml
Microplate
1 ml
150 µl
1 ml
150 µl
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5.
Incubate at room temperature for at least
5 min. Samples should not be incubated
longer than 1 hr at room temperature.
6.
Set the spectrophotometer to 595 nm. Zero
the instrument with the blank sample (not
required for microplate readers). Measure the
absorbance of the standards, blanks, and
unknown samples. Refer to Section 3 for
data analysis.
Note: If the spectrophotometer has a
reference and sample holder, the instrument
can be zeroed with the blank samples. If the
effect of buffer on absorbance is required,
zero the instrument with a cuvette filled with
water and dye reagent in the reference
holder.
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Section 3
Data Analysis
1.
If the spectrophotometer or microplate reader
was not zeroed with the blank, then average
the blank values and subtract the average
blank value from the standard and unknown
sample values.
2.
Create a standard curve by plotting the
595 nm values (y-axis) versus their concentration in µg/ml (x-axis). Determine the unknown
sample concentration using the standard
curve. If the samples were diluted, adjust the
final concentration of the unknown samples
by multiplying by the dilution factor used.
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3.
The microplate procedure may yield lower
values than the standard and microassay
procedures due to a shorter light path used
in the microplate reader. This may decrease
the level of detection of the assay.
4.
Standard curve examples for the standard
5 ml procedure and the microassay
procedure are listed in Figures 1 and 2,
respectively.
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Fig 1. Typical standard curves using the standard 5 ml
procedure with BSA and gamma-globulin standards.
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Fig 2. Typical standard curves using the microassay
procedure with BSA and gamma-globulin standards.
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Section 4
FAQs and Troubleshooting
Questions
Recommendations
1 The buffer that I normally use is
not in the list of compatible
reagents. How will I know if it
interferes with the Quick Start
Bradford assay?
It is best to run two standard curves,
one with protein in the same buffer as
your sample and one with protein in
water, and then plot a graph of
protein concentration versus
absorbance. If the buffer does not
interfere, the two standard curves will
have identical slopes. Adding the
buffer or interfering component to the
standards used to construct the
standard curve for the actual protein
assay can compensate for partial
interference.
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2 My sample contains a
If the protein concentration is high
detergent concentration that is enough, a sample with detergent can
incompatible with the Quick
be diluted so that the concentration of
Start Bradford assay. How can I detergent is reduced to 0.1% or less.
assay for protein?
Alternatively, the Bio-Rad DC™ (detergent compatible) protein assay can be
used (catalog #500-0111). The DC
protein assay is a modified Lowry
assay, which works in the
presence of 1% ionic or nonionic
detergent.
3 Is any sample preparation
required?
In general, no. However, the protein
must be solubilized. The sample
cannot be a suspension or an
unfiltered homogenate.
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4 Does the binding of the blue
dye to cuvettes skew results?
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Page 30
Bio-Rad’s disposable polystyrene
cuvettes (catalog #223-9950) are
recommended for the protein assay.
When using quartz cuvettes, the
amount of dye that binds to them is
negligible (Bradford 1976). Therefore,
removal of the residual blue color
between each sample reading is
unnecessary. However, since the
cuvettes may be used for subsequent
procedures, there are several recommended treatments for dye removal:
•
•
•
24
Rinse the cuvette with methanol,
or
Rinse the cuvette with glassware
detergent, followed by ddi water,
or
Soak the cuvette in 0.1 N HCl for
a few hours, then wash with ddi
water.
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5 May I use a wavelength other
than 595 nm?
Yes. Absorbance can be measured at
580–610 nm.
6 Precipitation occurs in the
tubes.
The samples contain a detergent in
the buffer. Dilute the sample to reduce
the detergent level or dialyze the
samples.
7 Absorbance of protein standard The 1x dye reagent may be cold from
and samples is very low.
4°C storage. Warm the dye reagent to
ambient temperature. The 1x dye
reagent may be old. If it is over 1 year
old, replace the dye reagent.
8 Absorbance of standard is
acceptable, but absorbance of
samples is very low.
The samples may contain a substance that interferes with the
reaction, such as detergent or basic
solutions. Check the compatibility
guide (Table 1). Dilute the sample.
Ensure the standards are diluted in
the same buffer as the samples.
The molecular weight of the sample
protein may be low. The lower limit of
detection for this method is approximately 3,000–5,000 daltons.
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Section 5
Ordering Information
Catalog # Description
500-0201 Quick Start Bradford Protein Assay Kit 1, includes
1 L 1x dye reagent and 5 x 2 ml bovine serum
albumin standard at 2 mg/ml
500-0202 Quick Start Bradford Protein Assay Kit 2, includes
1x dye reagent (1 L), bovine serum albumin standard
set (2 sets of 7 standards, 0.125–2.0 mg/ml, 2 ml)
500-0203 Quick Start Bradford Protein Assay Kit 3, includes
1x dye reagent (1 L), bovine g-globulin standard
(5 x 2 mg/ml)
500-0204 Quick Start Bradford Protein Assay Kit 4, includes
1 L 1x dye reagent and bovine gamma globulin
standard set with 2 x 2 ml each concentration
500-0205 Quick Start Bradford 1 x Dye Reagent, 1L
500-0206 Quick Start Bovine Serum Albumin Standard,
2 mg/ml, 5 x 2 ml tubes
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Catalog # Description
500-0207 Quick Start Bovine Serum Albumin Standard Set,
2 x 2 ml each concentration, (2, 1.5, 1, 0.75, 0.5,
0.25, and 0.125 mg/ml)
500-0208 Quick Start Bovine Gamma Globulin Standard,
2 mg/ml, 5 x 2 ml tubes
500-0209 Quick Start Bovine Gamma Globulin Standard Set,
2 x 2 ml each concentration (2, 1.5, 1, 0.75, 0.5,
0.25, and 0.125 mg/ml)
223-9950 Disposable Polystyrene Cuvettes, 3.5 ml, box of 100
223-9955 Disposable Polystyrene Cuvettes, 1.5 ml, box of 100
224-0096 Costar 96-well Flat-Bottom EIA Plate, polystyrene,
5 per package, box of 100
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Section 6
References
Bradford MM, A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of
protein-dye binding, Anal Biochem, 72, 248–254 (1976)
Compton SJ and Jones CG, Mechanism of dye response and
interference in the Bradford protein assay, Anal Biochem 151,
369–374 (1985)
Fanger B, Adaptation of the Bradford protein assay to
membrane-bound proteins by solubilizing in glucopyranoside
detergents, Anal Biochem 162, 11–17 (1987)
Fazekas de St. Groth S et al., Two new staining procedures for
quantitative estimation of proteins on electrophoretic strips,
Biochim Biophys Acta 71, 377–391 (1963)
Reisner AH et al., The use of Coomassie Brilliant Blue G-250
perchloric acid solution for staining in electrophoresis and isoelectric
focusing on polyacrylamide gels, Anal Biochem 64, 509–516 (1975)
28
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Sedmak JJ and Grossberg SE, A rapid, sensitive and versatile
assay for protein using Coomassie Brilliant Blue G-250, Anal
Biochem 79, 544–552 (1977)
Spector T, Refinement of the Coomassie blue method of protein
quantitation. A simple and linear spectrophotometric assay for
less than or equal to 0.5 to 50 micrograms of protein, Anal
Biochem 86, 142–146 (1978)
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Section 7
Appendix
5 ml Standard Assay
Tube #
1
2
3
4
5
6
7
8 (blank)
Standard
Volume (µl)
300
375
325
175
325
325
325
–
Source of
Standard
2 mg/ml stock
2 mg/ml stock
2 mg/ml stock
Tube 2
Tube 3
Tube 5
Tube 6
–
Diluent
Final
Volume (µl) [Protein] (µg/ml)
0
2,000
125
1,500
325
1,000
175
750
325
500
325
250
325
125
325
0
1 ml Standard Assay
Tube #
1
2
3
4
5
6
7
8 (blank)
Standard
Volume (µl)
70
75
70
35
70
70
70
–
Source of
Standard
2 mg/ml stock
2 mg/ml stock
2 mg/ml stock
Tube 2
Tube 3
Tube 5
Tube 6
–
30
Diluent
Final
Volume (µl) [Protein] (µg/ml)
0
2,000
25
1,500
70
1,000
35
750
70
500
70
250
70
125
70
0
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Page 37
Microplate Standard Assay
Tube #
1
2
3
4
5
6
7
8 (blank)
Standard
Volume (µl)
20
30
20
20
20
20
20
–
Source of
Standard
2 mg/ml stock
2 mg/ml stock
2 mg/ml stock
Tube 2
Tube 3
Tube 5
Tube 6
–
Diluent
Final
Volume (µl) [Protein] (µg/ml)
0
2,000
10
1,500
20
1,000
20
750
20
500
20
250
20
125
20
0
2 ml Microassay Cuvette Standard Dilutions
Tube #
1
2
3
4
5
6
7
8 (blank)
Standard
Volume (µl)
40
65
30
3,250
3,250
3,250
3,000
–
Source of
Standard
2 mg/ml stock
2 mg/ml stock
2 mg/ml stock
Tube 2
Tube 4
Tube 5
Tube 6
–
31
Diluent
Final
Volume (µl) [Protein] (µg/ml)
3,160
25
6,435
20
3,970
15
3,250
10
3,250
5
3,250
2.5
3,000
1.25
3,200
0
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Page 38
2 ml Microassay Cuvette Dilutions for BSA or
Gamma-Gobulin Standard Sets
Tube #
1
2
3
4
5
6
7
8 (blank)
Standard
Volume (µl)
40
35
35
35
35
35
35
–
Source of
Standard
2 mg/ml
2 mg/ml
1.5 mg/ml
1 mg/ml
0.5 mg/ml
0.25 mg/ml
0.125 mg/ml
–
Diluent
Final
Volume (µl) [Protein] (µg/ml)
3,160
25
3,465
20
3,465
15
3,465
10
3,465
5
3,465
2.5
3,465
1.25
3,200
0
Microplate Microassay Dilutions for
2 mg/ml BSA or Gamma-Globulin
Tube #
1
2
3
4
5
6
7
8 (blank)
Standard
Volume (µl)
10
10
6
500
500
500
500
–
Source of
Standard
2 mg/ml stock
2 mg/ml stock
2 mg/ml stock
Tube 2
Tube 4
Tube 5
Tube 6
–
32
Diluent
Final
Volume (µl) [Protein] (µg/ml)
790
25
990
20
794
15
500
10
500
5
500
2.5
500
1.25
500
0
4110065A.qxp
9/25/2007
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Page 39
Microplate Microassay Dilutions for BSA or
Gamma-Globulin Standard Sets
Tube #
1
2
3
4
5
6
7
8 (blank)
Standard
Volume (µl)
10
5
5
5
5
5
5
–
Source of
Standard
2 mg/ml
2 mg/ml
1.5 mg/ml
1 mg/ml
0.5 mg/ml
0.25 mg/ml
0.125 mg/ml
–
33
Diluent
Final
Volume (µl) [Protein] (µg/ml)
790
25
495
20
495
15
495
10
495
5
495
2.5
495
1.25
500
0
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Page 2
Bio-Rad Laboratories, Inc.
2000 Alfred Nobel Dr.
Hercules, CA 94547 USA
(510) 741-1000
1-800-424-6723 US only
4110065 Rev A
BSA Standard
2 mg/mL
Tube
BSA Volume (uL) PBS Volume (uL)
Concentration (ug/uL)
Concentration (ug/mL)Absorbance
1 (BLANK)
0
24
0
0
0
2
2
22
0,166666667
166,6666667
1,11
3
4
20
0,333333333
333,3333333
1,33
4
6
18
0,5
500
2,09
5
8
16
0,666666667
666,6666667
3,14
6
10
14
0,833333333
833,3333333
3,28
7
12
12
1
1000
3,57
C1V1 = C2V2
C1 = 2mg/mL V1 = initial BSA volume
V2 = total final volume (BSA +PBS)
C2 = C1V1/V2
linear range for BSA is 125 ug/mL – 1000 ug/mL
1ug/uL=1000ug/mL
BSA STANDARD ABSORBANCE
4
3,5
3
2,5
Absorbance
2
1,5
1
0,5
0
0
200
400
600
Concentration(ug/mL)
800
Protein sample (unknown concentration)
Tube
Sample Volume (uL)PBS Volume (uL) Absorbance (y)
1 (BLANK)
0
24
0
2
2
22
1,01
3
4
20
1,87
4
6
18
2,24
5
8
16
2,5
6
10
14
3,26
7
12
12
3,47
y=ax+b
x=(y-b)/a
y=0.0033x+0.5
y is absorbance x=cuvette concentration in (ug/mL)
x=(y-0.5)/0.0033
C1V1 = C2V2
BSORBANCE
y = 0,0033x + 0,5
1000
1200
Cuvette Concentration
Sample
(ug/mL)
Concenration
Sample
(ug/mL)
Concenration (mg/mL)
154,5454545
415,1515152
527,2727273
606,0606061
836,3636364
900
1854,545455
2490,909091
2109,090909
1818,181818
20007,27273
1800
1,854545455
2,490909091
2,109090909
1,818181818
2,007272727
1,8
2,045090909
Hussain Shahani
MCB 253 Section F
10 Feb 2021
Quantification Protocol: Bradford Assay
Background of Bradford Assay
In 1976 Marion M Bradford developed the Bradford protein assay. It aims to measure
the protein concentration in a solution by spectroscopic analysis. As in all protein tests the
reaction is dependent on the amount of amino acid composition present in a protein sample.
The preference of this assay is due to its quickness and accuracy. When compared to other
methods of estimating protein concentration, it takes 5 minutes. However, underestimation may
occur for protein with low arginine content. For instance, the Bradford assay has much more
sensitivity to bovine serum albumin (BSA) than immunoglobulin G (IgG). So, it is likely for
an investigator to underestimate protein amount with BSA.
It is essential to have a right amount of protein sample since the concentration of amino
acids in different proteins presents sensitivity of colorimetric assays. It also uses similar amount
of proteins as in the Lowry assay. The range provide in Bradford assay for better accuracy is 1
to 20 micrograms and 200 micrograms in the extreme. The concentration volume range is 1ml
to 5.5ml. The dye reagent, dye-albumin complex solution, binds with the present protein
resulting to a shift in absorption of dye from 465-595 nanometres (QuickStartTM Bradford
Protein Assay Instruction Manual). Notably, there are three forms of the dye, cationic (red),
anionic (blue) and neutral (green). The spectrophotometer is monitored to records the
absorption where the blue coloured proteins are identified. To calculate the concentration of
the unknown protein, we will plot the concentration/absorbance graph of the protein. We will
then compare its values with the value of BSA standard.
Materials
➢ Protein Sample
➢ Coomassie Blue Stain (Bio-Rad 161-0786)
➢ Bradford Reagent –
➢ UV-Vis spectrophotometer
➢ BSA Standard (2 mg/ml) (Bio-Rad 500-0206)
➢ 1.5 ml Semi-Micro Cuvettes
➢ Microcentrifuge
➢ Microfuge Tubes
➢ 1X PBS
➢ Vortex
➢ Microfuge Tubes
➢ P20, P200, and P1000 Pipettes with Tips
Procedure
The Bradford reagent should be light brown in colour, so filtration is required to get rid of the
blue components. Alternatively, homemade reagent can be used which are quite less
expensive but not effective like the Bio-Rad product.
1. Gently warm up the spectrophotometer at room temperature before use.
2. Prepare standard and unknown dilute solutions. We will prepare 6 dilutions for the
protein BSA with a known concentration of 2mg/ml.We will also have a blank
cuvette. So, in total we will have 7 cuvettes including a blank and 6 protein samples.
3. Use the P20 pipette to take out 20-µl of each dilution and place them into separate 1.2
ml cuvettes. The cuvettes will be labelled as followed BSA 1-BSA6 including a
blank. Each sample will contain 1.2ml Bradford reagent and 24ul protein dilution
(including the buffer solution)
4. Set the Spectrophotometer to 595 nm and warm up the Spectrophotometer
5. Now that the Spectrophotometer is warmed up, put the blank in the spectrophotometer
to zero it.
6. Measure the absorbance at 595 nm of the standards and unknowns and record them in
the data table below.
7. Plot the Standard Curve using the absorbance and protein concentration.
8. Make a line of best fit and use the equation to calculate your unknown’s concentration
Table Result
BSA
2 mg/mL
Standard
Tube
BSA
PBS
Concentration
Concentration
Volume
Volume
(ug/uL)
(ug/mL)
(uL)
(uL)
1 (BLANK)
0
24
0
0
2
2
22
0.166666667
166.6666667
3
4
20
0.333333333
333.3333333
4
6
18
0.5
500
5
8
16
0.666666667
666.6666667
6
10
14
0.833333333
833.3333333
7
12
12
1
1000
C1V1 = C2V2
C1 = 2mg/mL
V1 = initial BSA volume
V2 = total final volume (BSA +PBS)
C2 = C1V1/V2
linear range for BSA is 125 ug/mL – 1000
ug/mL
1ug/uL=1000ug/mL
Explanation of results:
It was expected that the values for the absorbance to be similar to the values of the BSA
standard due to the liner relation between concentration and absorbance. We will be able to
compare the concentration of BSA to the unknown proteins. In this experiment various
proteins concentrations were calculated, and absorbance was measured at 595m using Beer’s
law. The results of this experiment concluded that at higher protein concentration, the
absorbance value was higher and when protein concentrations were low, the absorbance
values were also low.
References
Bradford, MM. A rapid and sensitive for the quantitation of microgram quantitites of protein
utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254. 1976.
General Reference: Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 5069 (1990).
Bio-Rad Company. “Quick Start Bradford Protein Assay: Instruction Manual.”
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