Bradford
protein assay
Considerations for use
The Bradford assay is very fast and uses about the same amount of protein as the Lowry assay. It
is fairly accurate and samples that are out of range can be retested within minutes.
The Bradford is recommended for general use, especially for determining protein content of cell
fractions and assesing protein concentrations for gel electrophoresis.
Assay materials including color reagent, protein standard, and instruction booklet are available
from Bio-Rad Corporation. The method described below is for a 100 µl sample volume using 5 ml
color reagent. It is sensitive to about 5 to 200 micrograms protein, depending on the dye quality.
In assays using 5 ml color reagent prepared in lab, the sensitive range is closer to 5 to 100 µg
protein. Scale down the volume for the "microassay procedure," which uses 1 ml cuvettes.
Protocols, including use of microtiter plates are described in the flyer that comes with the Bio-
Rad kit.
Principle
The assay is based on the observation that the absorbance maximum for an acidic solution
of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs.
Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible
color change. The assay is useful since the extinction coefficient of a dye-albumin complex
solution is constant over a 10-fold concentration range.
Equipment
In addition to standard liquid handling supplies a visible light spectrophotometer is needed, with
maximum transmission in the region of 595 nm, on the border of the visible spectrum (no special
lamp or filter usually needed). Glass or polystyrene (cheap) cuvettes may be used, however the
color reagent stains both. Disposable cuvettes are recommended.
Procedure
Reagents
1. Bradford reagent: Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml
95% ethanol, add 100 ml 85% (w/v) phosphoric acid. Dilute to 1 liter when the dye has
completely dissolved, and filter through Whatman #1 paper just before use.
2. (Optional) 1 M NaOH (to be used if samples are not readily soluble in the color reagent).
The Bradford reagent should be a light brown in color. Filtration may have to be repeated to rid
the reagent of blue components. The Bio-Rad concentrate is expensive, but the lots of dye used
have apparently been screened for maximum effectiveness. "Homemade" reagent works quite
well but is usually not as sensitive as the Bio-Rad product.
Assay
1. Warm up the spectrophotometer before use.
2. Dilute unknowns if necessary to obtain between 5 and 100 µg protein in at least one assay
tube containing 100 µl sample
3. If desired, add an equal volume of 1 M NaOH to each sample and vortex (see Comments
below). Add NaOH to standards as well if this option is used.
4. Prepare standards containing a range of 5 to 100 micrograms protein (albumin or gamma
globulin are recommended) in 100 µl volume. See how to set up an assay for suggestions
as to setting up the standards.
5. Add 5 ml dye reagent and incubate 5 min.
6. Measure the absorbance at 595 nm.
Analysis
Prepare a standard curve of absorbance versus micrograms protein and determine amounts
from the curve. Determine concentrations of original samples from the amount protein,
volume/sample, and dilution factor, if any.
Comments
The dye reagent reacts primarily with arginine residues and less so with histidine, lysine,
tyrosine, tryptophan, and phenylalanine residues. Obviously, the assay is less accurate for basic
or acidic proteins. The Bradford assay is rather sensitive to bovine serum albumin, more so than
"average" proteins, by about a factor of two. Immunoglogin G (IgG - gamma globulin) is the
preferred protein standard. The addition of 1 M NaOH was suggested by Stoscheck (1990) to
allow the solubilization of membrane proteins and reduce the protein-to-protein variation in
color yield.
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.
Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990).
Bicinchoninic Acid (BCA) Protein Assay (Smith)
Considerations for use
The bicinchoninic acid (BCA) assay is available in kit form from Pierce (Rockford, Ill.). This
procedure is very applicable to microtiter plate methods. The BCA is used for the same reasons
the Lowry is used. Stoscheck (1990) has suggested that the BCA assay will replace the Lowry
because it requires a single step, and the color reagent is stable under alkaline conditions.
Both a standard assay for concentrated proteins and a micro assay for dilute protein solutions
are described below.
Principle
BCA serves the purpose of the Folin reagent in the Lowry assay, namely to react with complexes
between copper ions and peptide bonds to produce a purple end product. The advantage
of BCA is that the reagent is fairly stable under alkaline conditions, and can be included in the
copper solution to allow a one step procedure. A molybdenum/tungsten blue product is
produced as with the Lowry.
Equipment
In addition to standard liquid handling supplies a visible light spectrophotometer is needed with
transmission set to 562 nm. Glass or polystyrene (cheap) cuvettes may be used.
Procedure 1 (standard assay)
Reagents
1. Reagent A: 1 gm sodium bicinchoninate (BCA), 2 gm sodium carbonate, 0.16 gm
sodium tartrate, 0.4 gm NaOH, and 0.95 gm sodium bicarbonate, brought to 100 ml with
distilled water. Adjust the pH to 11.25 with 10 M NaOH.
2. Reagent B: 0.4 gm cupric sulfate (5 x hydrated) in 10 ml distilled water.
3. Standard working solution (SWR): Mix 100 volumes reagent A with 2 volumes reagent B.
4. The stock solutions are stable. The working solution is stable for 1 week and should be
green.
Assay
1. Prepare samples containing 0.2 to 50 micrograms protein in microliters.
2. Add 1 ml SWR to each 20 microliters sample and mix. Incubate 30 min. at 60 degrees C.
3. Cool the samples and read at 562 nm. Color will be stable for at least one hour.
Procedure 2 (micro assay)
Reagents
1. Reagent A: 8 gm sodium carbonate monohydrate, 1.6 gm sodium tartrate, brought to 100
ml with distilled water. Adjust the pH to 11.25 with 10 M NaOH.
2. Reagent B: 4 gm BCA in 100 ml distilled water.
3. Reagent C: 0.4 gm cupric sulfate (5 x hydrated) in 10 ml water.
4. Working solution: Mix 1 volume reagent C with 25 volumes reagent B, then add 26
volumes reagent A to the C/B mixture.
Assay
1. Prepare samples containing 0.2 to 50 micrograms protein in 500 microliters.
2. Add 500 microliters working solution to each 500 microliters sample and mix. Incubate
60 min. at 60 degrees C.
3.
4. Cool the samples and read at 562 nm.
Analysis
Prepare a standard curve of absorbance versus micrograms protein (or vice versa), and
determine amounts from the curve. Determine concentrations of original samples from the
amount protein, volume/sample, and dilution factor, if any. If you are unfamiliar with how to
obtain a protein concentration for a diluted sample from a standard curve, see how to prepare
and use a protein standard curve.
Comments
A longer incubation increases the sensitivity of the assay. The heating can be stopped earlier to
prevent the color from becoming too dark. The assay can be performed at room temperature, but
there is greater variability among proteins and the assay is less sensitive.
Reference
Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990).
