Bicinchoninic Acid Protein Assay Kit Catalog Numbers BCA1 AND B9643
TECHNICAL BULLETIN
Synonym: BCA
Product Description
Protein determination is one of the most common operations performed in biochemical research. The
principle of the bicinchoninic acid (BCA) assay is similar to the Lowry procedure,1in that both rely on the formation of a Cu 2+-protein complex under alkaline conditions, followed by reduction of the Cu 2+to Cu 1+. The amount of reduction is proportional to the protein present. It has been shown that cysteine, cystine,
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tryptophan, tyrosine, and the peptide bond 2are able to
reduce Cu 2+to Cu 1+
.BCA forms a purple-blue complex with Cu 1+in alkaline environments, thus providing a
basis to monitor the reduction of alkaline Cu 2+
by
proteins.
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The BCA assay is more sensitive and applicable than either biuret or Lowry procedures. In addition, it has less variability than the Bradford assay. The BCA assay has many advantages over other protein determination techniques: • It is easy to use.
• The color complex is stable.
• There is less susceptibility to detergents.•
It is applicable over a broad range of protein concentrations.
In addition to protein determination in solution, the BCA protein assay has other applications, including
determination of protein covalently bound to agarose supports and protein adsorbed to multiwell plates. There are two distinct ways to perform a protein assay. A protein assay can be set up to measure the
concentration of the unknown protein sample (mg/ml), or it can be set up to determine the total amount of protein in the unknown protein sample (mg). The BCA assay has a linear concentration range between 200–1,000 µg of protein per milliliter. In the standard assay, only 0.1 ml protein sample is used, so the assay has a total linear protein range of 20–100 µg.
Reagents
Bicinchoninic Acid Solution, Catalog Number B9643Reagent A is a 1,000 ml solution containing
bicinchoninic acid, sodium carbonate, sodium tartrate, and sodium bicarbonate in 0.1 N NaOH (final pH 11.25).
Copper(II) Sulfate Pentahydrate 4% Solution, Catalog Number C2284
Reagent B is a 25 ml solution containing 4% (w/v) copper(II) sulfate pentahydrate.
Protein Standard (Bovine Serum Albumin -BSA) Solution, Catalog Number P0914
This product is supplied in 5 flame-sealed glass
ampules, each containing 1.0 ml of a solution consisting of 1.0 mg/ml bovine serum albumin in 0.15 M NaCl with 0.05% sodium azide as a preservative.
Materials required depending on assay format used but not provided
• Spectrophotometer capable of accurately measuring absorbance in the 560 nm region.• 96 well plates, Catalog Number M0156
• 96 well plate sealing film, Catalog Number Z369667• Test tubes, 13 ×100 mm,
Catalog Number CLS980013
• 1 ml Disposable Plastic Cuvettes,
Catalog Number C5416Precautions and Disclaimer
This product is for R&D use only, not for drug,
household, or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.
Preparation Instructions
The BCA Working Reagent is prepared by mixing
50 parts of Reagent A with 1 part of Reagent B. Mix the BCA Working Reagent until it is light green in color.
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Storage/Stability
Store Reagents A and B at room temperature. Reagent A, without Reagent B added, is stable for at least one year at room temperature in a closed container. The BCA Working Reagent (Reagent A mixed with Reagent B) is stable for one day.
Store the Protein Standard at 2–8 °C.
Procedure
In the standard assay, 20 parts of the BCA Working Reagent are then mixed with 1 part of a protein sample. For the 96 well plate assay, 8 parts of the BCA Working Reagent are mixed with 1 part of a protein sample. The sample is either a blank, a BSA protein standard, or an unknown sample. The blank consists of buffer with no protein. The BSA protein standard consists of a known concentration of bovine serum albumin, and the unknown sample is the solution to be assayed.
BCA assays are routinely performed at 37 °C. Color development begins immediately and can be accelerated by incubation at higher temperatures. Higher temperatures and/or longer incubation times can be used for increased sensitivity. Incubation at lower temperatures can slow down color development (see Procedures A and B). The absorbance at 562 nm is recorded and the protein concentration is determined by comparison to a standard curve.
A.Standard 2.1 ml Assay Protocol
(Linear concentration range is 200-1,000 µg/ml or
20-100 µg of total protein)
This is the standard assay that can be performed in a test tube. This procedure uses 0.1 ml of a protein sample and 2 ml of the prepared BCA Working Reagent. The instructions are a step-by-step procedure on how to perform the standard assay. If a nonstandard assay is used (96 well plate) adjust the volumes accordingly.
Note: It is necessary to create a standard curve during each assay, regardless of the format used.1.Prepare the required amount of BCA Working
Reagent needed for the assays (see Table 1). The final volume used in the assay depends upon the
application and the equipment available. Table 1
can be used to determine the volume of BCA
Working Reagent to prepare, depending on how
many blanks, BSA protein standards, and unknown samples are to be assayed. Combine the volumes
苏打绿主唱是谁of Reagents A and B specified in the table. Mix until the BCA Working Reagent is a uniform, light green color.
Table 1.
Volume of BCA Working Reagent to prepare. This is dependent on how many blanks, BSA protein standards, and unknown samples are to be assayed.
2.Prepare standards of different concentrations.
These BSA protein standards can range from
200–1,000 µg/ml (20–100 µg of total protein). This is accomplished by making serial dilutions starting
from the 1 mg/ml standard, and then using 0.1 ml of each diluted standard in the assay. It is best to
make the dilutions in the same buffer as the
unknown sample (see Table 2). Deionized water
may be used as a substitute for the buffer, but any interference due to the buffer will not be
compensated for in the BSA protein standards.
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Table 2
EXAMPLE of Standard Assay Set Up Table
For protein samples with unknown concentrations, it may be necessary to prepare a dilution scheme to ensure the concentration is within the linear range of 200–1,000 µg/ml. Two different unknown sa
mples are represented in Table 2 by tubes 7 and 8. Tube 7 is an unknown sample with a 5-fold dilution, while tube 8 is a different unknown sample at a 10-fold dilution. Researchers must determine their own dilution schemes based on their estimation of the concentration of each unknown sample.
3.Add 2 ml of the BCA Working Reagent to 0.1 ml of
each BSA protein standard, blank, and unknown
sample. Vortex gently for thorough mixing. The total liquid volume in the test tube is 2.1 ml.
4.The following incubation parameters may be used:
60 °C for 15 minutes Or
37 °C for 30 minutes Or
25 °C (Room Temperature) from 2 hours to
overnight
5.If required, allow the tubes to cool to room
temperature.
绿的旋律6.Transfer the reaction solutions into a cuvette.
7.Measure the absorbance of the solution at 562 nm.
Color development continues slowly after cooling to room temperature, but no significant error is seen if all the tubes are read within 10 minutes of each
other. Create an assay table as needed and a
standard curve based on either the BSA protein
standard concentration or on the amount of protein present in the BSA protein standard (Examples are shown in the results).8.Determine protein concentration by comparison of
the absorbance of the unknown samples to the
standard curve prepared using the BSA protein
standards.
Results Based on the Standard Assay
Create a table with the absorbance results obtained during the assay. A separate standard curve should be generated for each assay performed. The amount of protein for tubes 1–6 was obtained from the known amount of BSA protein standard added.
Note: The data below should not be used as a replacement of a standard curve. The absorbance of the BSA protein standards (tubes 1–6) in each assay will differ from those presented here. The amount of protein recorded for tubes 7 and 8 was obtained from the standard curve.
Table 3.
EXAMPLE of Assay Data Table
After obtaining the results, create a standard curve to determine the protein concentration in the unknown sample. Plot the Net Absorbance at 562 nm versus the BSA protein standard concentrations
(µg/ml, Tubes 1–6).
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Graph 1.
Standard Curve produced from Assay Data
The standard curve indicates the unknown protein sample in test tube 7 (Net A562= 0.542) contains
700 µg/ml of protein.
The actual concentration of protein present in the unknown sample is calculated as follows:
(µg/ml of unknown protein sample) times
(Dilution Factor)
(700 µg/ml) ×(5) = 3,500 µg/ml of protein
B.96 Well Plate Assay
(Linear concentration range is 200-1,000 µg/ml or
5-25 µg of total protein)
The BCA assay can be adapted for use in 96 well plates. These plates can be used as long as five main points remain unchanged:
1.Read the absorbance at 562 nm. For a plate
reader, which does not have the exact wavelength filter, a filter in the range of 540-590 nm can be
substituted.
2.The ratio of BCA Working Reagent to protein
sample will have to be modified from the Standard Assay.
Examples:
Standard Assay-(Test Tube): 0.10 ml protein
sample to 2 ml BCA Working Reagent (1:20)
96 well plate-25 µl protein sample to 200 µl BCA
Working Reagent (1:8). When using multiwell
plates, make sure the unknown samples, blanks, or standards are present in the wells prior to adding
the BCA Working Reagent to facilitate mixing.3.Make sure the protein assay containers are sealed
(cover the plates with film) and incubate the
samples for:
60 °C for 15 minutes Or
37 °C for 30 minutes Or
f4流星雨mv25 °C (Room Temperature) from 2 hours to
overnight
4.Keep the protein sample concentration between
200–1,000 µg/ml (5–25 µg total protein).
5. A separate standard curve will have to be
determined for each assay protocol. The pathlength in each assay is dependent on the assay container (cuvettes or multiwell plates) and/or the reaction
volume. These and others changes like the BCA
Working Reagent to protein sample ratio affect the Net Absorbance values.
一起摇摆C.TCA Concentration-BCA Assay Protocol
By using this procedure it is possible to remove some of the interfering substances that are described in the compatibility chart. It is also possible to increase the concentration of the unknown sample using this procedure.
1.Add the unknown samples and BSA protein
standards to separate microcentrifuge tubes and
adjust the final volumes to 1 ml with deionized
water. Larger volumes can also be used by
adjusting the following volumes accordingly.
2.Add 0.1 ml of a 0.15% (w/v) solution of sodium
deoxycholate (Catalog Number D5670) prepared
with deionized water.
3.Mix and let stand for 10 minutes at room
temperature. It is also acceptable to let stand on ice for 10 minutes.
4.Add 0.1 ml of 6.1 N (∼100% w/v) solution of
trichloroacetic acid (TCA, Catalog Number T0699).
5.Cap and vortex each sample.
6.Incubate for 5 minutes at room temperature. It is
also possible to let stand on ice for 5 minutes.蝴蝶是谁
7.Centrifuge the samples for 15 minutes at room
temperature in a microcentrifuge at full speed.
8.Carefully decant or pipette the supernatant of each
sample. Do not disturb the pellet.
5 9.Solubilize each pellet by adding 0.04 ml of a
5% (w/v) solution of sodium dodecyl sulfate (SDS,
Catalog Number L6026) prepared with a 0.1 N
sodium hydroxide solution (Catalog Number
72076). Mix well until the pellet is completely
dissolved.
10.Pipette 0.06 ml of deionized water into the tube to
bring the sample volume to 0.10 ml, which can then
be used in the standard 2.1 ml assay procedure. It
is possible to add less water if a smaller volume
assay is to be performed.
11.Vortex each sample and proceed onto the 2.1 ml
standard assay protocol or a custom assay.
Compatibility Chart
The amount listed is the maximum amount of material
allowed in the protein sample without causing a
noticeable interference.
Incompatible Substances
Amount Compatible
Buffer Systems
N-Acetylglucosamine (10 mM)in
PBS, pH 7.2
10 mM ACES, pH 7.825 mM Bicine, pH 8.4 20 mM Bis-Tris, pH 6.533 mM
CelLytic™B Reagent
undiluted no interference
Calcium chloride in TBS, pH 7.210 mM CHES, pH 9.0100 mM Cobalt chloride in TBS, pH 7.20.8 M E
PPS, pH 8.0100 mM Ferric chloride in TBS, pH 7.210 mM HEPES100 mM MOPS, pH 7.2100 mM Nickel chloride in TBS10 mM
PBS; Phosphate (0.1 M), NaCl (0.15 M), pH 7.2
undiluted no interference
PIPES, pH 6.8100 mM Sodium acetate, pH 4.8200 mM Sodium citrate, pH 4.8 or pH 6.4200 mM Tricine, pH 8.025 mM Triethanolamine, pH 7.825 mM Tris250 mM
TBS; Tris (25 mM), NaCl (0.15 M), pH 7.6 (Catalog Number T5030)
undiluted no interference
Tris (25 mM), Glycine (1.92 M), SDS (0.1%), pH 8.3 (Catalog Number T4904)
undiluted no interference
Zinc chloride (10 mM) in TBS, pH 7.210 mM
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