Download BCA Total protein colorimetric assay

bicinchoninic Protein Assay Kit
eMP014500 QuantumProtein Sufficient for 500 test tubes or 5000
microplate assays
Reagent A – 2 x 500 ml (bicinchoninic acid salt, sodium carbonate, sodium
bicarbonate and sodium tartrate, in 0,1 M naoH)
Reagent b – 25 ml (4% aqueous solution of CuSo4 5H2o)
For Research Use Only
Storage and Stability
12 months at room temperature
Introduction
QuantumProtein bicinchoninic Acid Protein Assay1,2,3 Kit is formulated for the colorimetric detection and quantitation
of total protein. Like the Lowry method, the assay relies on the reduction of Cu2+ ions by protein. the Cu+ thus formed are
detected by conversion into a violet-coloured substance by reaction with bicinchoninate. the absorbance at 562 nm of
the Cu(I)-(bicinchoninate)2 complex is directly proportional to protein concentration over a broad working range (20-2,000
µg/mL). the bicinchoninic Acid Protein Assay is more sensitive than the Lowry method and less subject to interference. In
particular, it is insensitive to detergents such as triton X-100 and SDS (5%).
IMPORTANT NOTES
• Precipitates which may form in Reagent A or b during storage should be dissolved by gentle warming and stirring.
• Reagents that chelate metal ions, change the pH of the assay or reduce copper are known to interfere with the assay. Please
check that the following components are not in the sample buffer: ascorbic acid, catecholamines, creatinine, cysteine, EGtA,
impure glycerol, hydrogen peroxide, hydrazides, iron, lipids, melibiose, phenol red, impure sucrose, tryptophan, tyrosine, uric
acid.
• other substances affect the assay to a lesser extent and, if their concentration in the sample buffer is below a certain value,
they can be tolerated. Please refer to table 2 for maximum compatible concentrations of many of these substances.
• It is necessary to create a standard curve during each assay regardless of the format used.
• QuantumProtein working solution is stable for several days at Rt. If not used immediately, it should be stored at room
temperature in a closed container
Materials Required
Equipment Required
• Spectrophotometer capable of measuring absorbance in the region 560 nm
• test tubes or 96 well plate
• Protein Standard
Working Solutions
Working Reagent: Mix 50 parts of reagent A and 1 part of Reagent b. the amount of Working Reagent required for
each sample is 2.0 ml for the test tube Procedure and 200 µl for the Microassay Plate Procedure. Prepare sufficient volume
of Working Reagent for the samples to be assayed plus the calibration standards. For example, for the standard test-tube
procedure with 9 standards (including a blank), 3 unknowns and 2 replicates for each sample 48 ml of Working Reagent
are required.
the Working Reagent is stable for several days when stored at room temperature in a closed container.
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Calibration Standards: Prepare a fresh set of protein standards in the 20-2,000 µg/ml range, preferably using the same diluent
as your sample. While the most accurate results are obtained using a pure sample of the protein to be measured as standard,
in many cases this is expensive or not available. therefore, the standards are generally prepared from a 1.0-2.0 mg/mL stock
solution of bovine Serum Albumin (eMR086050).
For several proteins a Correction Factor is reported in table 1.
table 1
Protein
Correction
Factor
Protein
Correction
Factor
Albumin, bovine serum
1.00
IgG, mouse
1.18
Aldolase, rabbit muscle
0.85
IgG, rabbit
1.12
α-Chymotrypsinogen, bovine
1.14
IgG, sheep
1.17
Cytochrome c, horse heart
0.83
Insulin, bovine pancreas
1.08
γ-globulin, bovine
1.11
Myoglobin, horse heart
0.74
IgG, bovine
1.21
ovalbumin
0.93
IgG, human
1.09
transferrin, human
0.89
Average Correction Factor ± St.Dev: 1.02 ± 0.15
Test Tube Procedure
the test tube procedure requires a larger volume (0,1 ml) of protein sample but, since the sample to working reagent ratio
is 1/20, the effect of interfering substances is minimized.
Step 1: Pipette 0,1 ml of each standard (including a blank) and unknown sample into a labelled test tube.
Step 2: Add 2 ml of Working Reagent to each tube and mix thoroughly.
Step 3: Cover the tubes and incubate at selected temperature and time. Heat the tubes in a water bath.
· Standard Protocol: 37°C for 30 minutes (working range = 20-2,000 µg/ml)
· Rt Protocol: Rt for 2 hours (working range = 20-2,000 µg/ml)
Step 4: Cool the tubes to Rt.
Step 5: Measure the absorbance at 562 nm of all the samples. Please note that even at Rt the color development continues:
no significant error will be introduced if the 562 nm absorbance measurements of all tubes are made within 10 minutes
Step 6: Subtract the 562 nm absorbance value of the blank from the readings of the standards and the unknowns.
Step 7: Plot the blank-corrected 562 nm reading for each standard vs. its concentration. Determine the protein concentration
of each unknown from the Calibration Plot.
Microplate Procedure
the Microplate Procedure requires a smaller volume (10-25 μl) of protein sample but, since the sample to Working
Reagent ratio is 1/8, it offers less flexibility in overcoming interfering substance concentrations and obtaining low level of
detection.
the working range is 20-2.000µg/ml
Step 1: Pipette 25 μl of each standard (including a blank) and unknown sample into a microwell plate.
Step 2: Add 200 μl of Working Reagent to each well and mix thoroughly.
Step 3: Cover the plate and incubate at 37°C for 30 minutes.
Step 4: Cool the plate to Rt.
Step 5: Measure the absorbance at 562 nm of all the samples on a plate reader.
Step 6: Subtract the 562 nm absorbance value of the blank from the readings of the standards and the unknowns.
Step 7: Plot the blank-corrected 562 nm reading for each standard vs. its concentration. Determine the protein concentration
of each unknown from the Calibration Plot.
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Troubleshooting Guide
NO COLOR DEVELOPMENT
Possible Cause
Precautions/Remedies
Chelating agents are present in the sample buffer
· Dialyze or desalt the sample.
· Dilute the sample.
SAMPLE COLOR IS LESS INTENSE THAN EXPECTED
Possible Cause
Precautions/Remedies
pH is altered by strong acid or alkaline buffer
· Dialyze or desalt the sample.
· Dilute the sample.
SAMPLE COLOR IS DARKER THAN EXPECTED
Protein concentration is too high
Dilute the sample.
Lipids or lipoproteins are present in the sample buffer
Add 2% SDS to the sample to eliminate interference
from lipids.
ALL THE TUBES ARE DARK PURPLE
Possible Cause
Precautions/Remedies
Reducing agents are present in the sample buffer
Dialyze or dilute the sample.
thiols are present in the sample buffer
Dialyze or dilute the sample.
Table 2 Compatibility Chart for the Standard test tube protocol
SubStAnCE
AMount
CoMPAtIble
Salts/Buffers
SubStAnCE
AMount
CoMPAtIble
MeS, pH 6.1
100 mM
ACeS, pH 7.8
25 mM
MeS (0.1 M), naCl (0.9%), pH 4.7
undiluted
Ammonium sulfate
1.5 M
MoPS, pH 7.2
100 mM
Asparagine
1 mM
Modified Dulbecco’s PbS, pH 7.4
undiluted
bicine, pH 8.4
20 mM
nickel chloride in tbS, pH 7.2
bis-tris, pH 6.5
33 mM
PbS Phosphate (0.1 M), naCl (0.15 M), pH
7.2
undiluted
Calcium chloride in tbS, pH 7.2
10 mM
PIPeS, pH 6.8
100 mM
RIPA lysis buffer; 50 mM tris, 150 mM
naCl, 0.5% DoC, 1% nP-40, 0.1% SDS,
pH 8.0
undiluted
Sodium acetate, pH 4.8
200 mM
na-Carbonate/na-bicarbonate (0.2 M), pH
9.4
undiluted
Cesium bicarbonate
100 mM
CHeS, pH 9.0
100 mM
na-Citrate (0.6 M), na-Carbonate (0.1 M),
pH 9.0
1:8 dilution*
na-Citrate (0.6 M), MoPS (0.1 M), pH 7.5
1:8 dilution*
Cobalt chloride in tbS, pH 7.2
0.8 mM
ePPS, pH 8.0
100 mM
Ferric chloride in tbS, pH 7.2
10 mM
Glycine•HCl pH 2.8
100 mM
Guanidine•HCl
4M
HePeS, pH 7.5
100 mM
Imidazole, pH 7.0
50 mM
Sodium azide
Sodium bicarbonate
Sodium chloride
10 mM
0.2%
100 mM
1M
Sodium citrate, pH 4.8 (or pH 6.4)
200 mM
Sodium phosphate
100 mM
tricine, pH 8.0
25 mM
triethanolamine, pH 7.8
25 mM
tris
250 mM
tbS; tris (25 mM), naCl (0.15 M), pH 7.6
undiluted
tris (25 mM), Glycine (192 mM), pH 8.0
1:3 dilution*
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Substance
Tris (25 mM), Glycine (192 mM), SDS
(0.1%), pH 8.3
Zinc chloride in TBS, pH 7.2
Amount
Compatible
undiluted
10 mM
Detergents
Substance
Amount
Compatible
Glucose
10 mM
2-Mercaptoethanol
0.01%
Thimerosal
0.01%
Brij®-35
5.0%
Misc. Reagents & Solvents
Brij®-56, Brij®-58
1.0%
Acetone
10%
CHAPS CHAPSO
5.0%
Acetonitrile
10%
Deoxycholic acid
5.0%
Aprotinin
Nonidet P-40 (NP-40)
5.0%
DMF, DMSO
10%
Octyl ß-glucoside
5.0%
Ethanol
10%
Octyl ß -thioglucopyranoside
5.0%
Glycerol (Fresh)
10%
SDS
5.0%
Hydrazide
NC
Span® 20
1.0%
Hydrides (Na2BH4 or NaCNBH3)
NC
Triton® X-100
5.0%
Hydrochloric Acid
100 mM
Triton® X-114
1.0%
Leupeptin
10 mg/L
Triton® X-305, X-405
1.0%
Methanol
10%
Tween®-20, Tween®-80
5.0%
Phenol Red
Tween®-60
Zwittergent® 3-14
5%
1.0%
Chelating agents
10 mg/L
PMSF
NC
1 mM
Sodium Hydroxide
Sucrose
100 mM
40%
EDTA
10 mM
TLCK
0.1 mg/L
EGTA
NC
TPCK
0.1 mg/L
200 mM
Urea
3M
Sodium citrate
Reducing & Thiol-Containing Agents
o-Vanadate (sodium salt), in PBS, pH 7.2
Ascorbic acid
NC
Cysteine
NC
Dithioerythritol (DTE)
1 mM
Dithiothreitol (DTT)
1 mM
ed2/0714/693_QuantumProtein
1 mM
* Diluted with ultrapure water
NC: not compatible
References
1. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner,
F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J.,
Klenk, D.C., Anal. Biochem. 1985; 150:76-85.
2. Walker, J.M., Methods Mol. Biol. 1994; 32:5-8.
3. Pingoud A., Urbanke, C, Hoggett J. Jeltsch, A., Biochemical
Methods, pp. 157-159. Wiley-VCH 2002.
MOLECULAR
BIOLOGY
EuroClone S.p.A.
Via Figino 20/22, 20016 Pero (MI) - Italy
Phone: +39.02.38195.1 - Fax +39.02.38101465
e-mail: [email protected]
www.euroclone.it
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