产品简要
公司名称 :
武汉博士德生物工程有限公司
产品类型 :
其他
产品名称 :
BCA蛋白分析体系
目录 :
AR0146
规格 :
1 kit
价格 :
50美元
更多信息或购买 :
文章摘录数: 3
参考文献 |
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图像
图像 1 :
Standard Curve of Net Absorbance versus protein sample concentration (Incubate at 37°C for 30 minutes).
产品信息
SKU号 :
AR0146
状态 :
Enabled
名称 :
BCA蛋白分析体系
目录名称 :
Western Blotting Reagents, Total Protein Analysis
价格 :
50美元
共轭标签 :
No
描述 :
BCA Protein Assay Kit, For quantitation of total protein
short description :
BCA Protein Assay Kit, Western Blotting Related Reagent
description1 :
List of Components
<tr style="font
weight:bold;"> Description Quantity Volume
Contents Catalog Number
BCA Reagent A 1 100mL containing
sodium carbonate, sodium bicarbonate, bicinchoninic acid and
sodium tartrate in 0.1M sodium hydroxide AR0146-
A
BCA Reagent B 1 5mL containing 4%
cupric sulfate AR0146-B
Albumin (BSA) Standards 1 20mL (2mg/mL)
containing bovine serum albumin (BSA) at 2mg/mL in 0.9%
saline and 0.05% sodium azide AR0146-C
Overview
Form Supplied
BCA Reagent A and B: ready-to-use 1X
solutions
. BSA Standard: stock solution for preparing a series of
standard protein dilutions
Assays per kit
Tube procedure: 50 assays
. Microplate procedure: 500 assays
Storage
Upon receipt store at room temperature. It is stable at room temperature for one year.
Note
If either Reagent A or Reagent B precipitates upon
shipping in cold weather or during long-term storage, dissolve
precipitates by gently warming and stirring solution. Discard any
kit reagent that shows discoloration or evidence of microbial
contamination.
Assay Range
20 - 2000ug/mL
Equivalent
Thermofisher (Product No. 23227), Assay Range: 20 -
2000ug/mL
. Millipore Sigma (Product No. BCA1), Assay Range: 200 -
1000ug/mL
Compatibility with reagents
Compatible with typical concentrations of most ionic
and nonionic detergents
. Incompatible with chelating agents, strong acids or bases, and
reducing agents: interfere with the reduction and chelating
reactions of the assay mechanism
Applications
Western blotting, protein expression assays, protein
profiling and characterization,
protein quantitation assays
. *Our Boster Guarantee covers the use of this product in the
above tested applications.
Physical State of A+B Reagent mixture
Light blue liquid
Reagent Color/Absorbance
Blue / A562 nm
Activity
Active in alkaline medium
Description
BCA Protein Assay Kit is a ready-to-use detergent-
compatible Western blot related total protein
analysis reagent used for the quick determination of total protein
concentration by measuring
absorbance at 562 nm and comparing to a protein standard
absorption vs. concentration curve,
according to Smith. The protein quantification process can be
finished in 45 minutes.
Cite This Product
BCA Protein Assay Kit (Boster Biological Technology,
Pleasanton CA, USA, Catalog # AR0146)
Assay Information
Assay Type Protein
Quantitative Measurement
Sample Type Serum, Plasma,
Cell culture extracts, Tissue Extracts
Technique Solution-based
detection of spectral absorbance at 562 nm; Smith Protein assay
Detection Method
Colorimetric/Spectroscopic
Equipment needed Test tubes
or miscroplates; Spectrophotometer; Microplate Reader
Assay Purpose Measure protein
concentration in solution
Incubation Requirements 30 min at
37°C
To be used with Lysates or homogenates,
prepared with non-reducing lysis and extraction buffers: Mammal
Tissue Protein Extraction Reagent, Mammal Cell Protein Extraction
Reagent, RIPA Lysis Buffer, Mitochondria Extraction Reagent,
Membrane Protein Extraction Kit, Cytoplasmic and Nuclear
Extraction Kit, Bacterial Cell Protein Extraction
Reagent
Assay Principle . The BCA Protein Assay is a detergent-compatible formulation
based on bicinchoninic acid (BCA) for the colorimetric detection
and quantization of total protein. This method combines the well-
known reduction of Cu2+ to Cu1+ by protein in an alkaline medium
(the biuret reaction) with the highly sensitive and selective
colorimetric detection of the cuprous cation (Cu1+) using a unique
reagent containing bicinchoninic acid. The purple-colored reaction
product of this assay is formed by the chelation of two molecules
of BCA with one cuprous ion. This water-soluble complex exhibits a
strong absorbance at 562 nm that is nearly linear with increasing
protein concentrations over a broad working range (20-2,000
ug/ml).
The BCA method is not a true end-point method; that is, the final
color continues to develop. However, following incubation, the
rate of continued color development is sufficiently slow to allow
large numbers of samples to be assayed together. The
macromolecular structure of protein, the number of peptide bonds
and the presence of four particular amino acids (cysteine,
cystine, tryptophan and tyrosine) are reported to be responsible
for color formation with BCA. Studies with di-, tri- and
tetrapeptides suggest that the extent of color formation caused by
more than the mere sum of individual color producing functional
groups.
Accordingly, protein concentrations generally are determined and
reported with reference to standards of a common protein such as
bovine serum albumin (BSA). A series of dilutions of known
concentration are prepared from the protein and assayed alongside
the unknown before the concentration of each unknown is determined
based on the standard curve. If precise quantization of an unknown
protein is required, it is advisable to select a protein standard
that is similar in quality to the unknown; for example, a bovine
gamma globulin (BGG) standard may be used when assaying
immunoglobulin samples. Two assay procedures are presented. Of
these, the Test Tube Procedure requires a larger volume (0.1 ml)
of protein sample; however, because it uses a sample to working
reagent ratio of 1:20 (v/v), the effect of interfering substances
is minimized.
Additional Materials Required . • 37°C water bath or 37°C incubator . • Microplate reader and spectrophotometer capable of accurately
measuring absorbance in the region of 540-595nm (562nm is the
optimal wavelength)
Important product information . 1. If this kit is received or stored cold, a precipitate may
form in Reagent A or Reagent B. To dissolve the precipitate, warm
the solution slowly at 37°C while mixing or microwave for a few
seconds. Discard the kit if it is contaminated by bacteria . 2. If interference caused by reducing substances or metal-
chelating substances contained in the sample remains, Bradford
Assay Kit is recommended. . 3. It is recommended that the standard of different
concentrations and samples be assayed in duplicate. Standard curve
should be plotted for each assay. . 4. Newly formed green turbidity will disappear after mixing
Reagent A and Reagent B thoroughly. It will not affect the
performance. . 5. Assayed sample amount will be reduced while using
spectrophotometer to detect protein concentration. When using 37°
C incubator, prevent the influence of water evaporation. . 6. Several ways for eliminating or minimizing the effects
of interfering substances:.
• Remove interfering substances by dialysis or gel filtration.
• Dilute the sample until substances no longer interfere.
• Since detergents of high concentrations also affect the results,
precipitate the proteins in the sample with trichloroacetic acid
(TCA). . 7.Avoid using substances including reducing substances,
chelating agents, strong acid and alkali since they interfere with
protein estimation even at very low concentration.
Assay Procedure . A. Test Tube Procedure . 1.Mix BCA Reagent A and BCA Reagent B at 50:1. i.e., mix 50 ml
of BCA Reagent A with 1 ml BCA Reagent B. Note: Use the following formula to determine the total volume
of working reagent required.
(# standards+ # unknowns) x (# replicates) x (volume of working
reagent per sample)= total volume of working reagent required. 2.Follow Table 1 to prepare a fresh set of standards. (Dilute
Albumin (BSA) Standards with 0.9% NaCl or PBS) . Table 1: Preparation of Albumin (BSA) Standards
Tube Number Volume of
Diluent (ul) Volume of BSA (ul) Final BSA
Concentration (ug/ml)
A 0 ul 900 ul of 2 mg/ml
Stock 2000 ug/ml
B 100 ul 300 ul of tube A 1500
ug/ml
C 300 ul 300 ul of tube A
1000 ug/ml
D 200 ul 200 ul of tube B
750 ug/ml
E 300 ul 300 ul of tube C
500 ug/ml
F 300 ul 300 ul of tube E
250 ug/ml
G 300 ul 300 ul of tube F
125 ug/ml
H 400 ul 100 ul of tube G
25 ug/ml
I 300 ul 0 0 (blank)
. 3.Add 0.1 ml of each standard and protein samples into separate
labeled test tubes. 4.Add 2 ml of BCA working reagent to each tube and mix well. 5.Incubate at 37°C for 30 minutes. Note: Increasing the incubation time and temperature can
increase the net 562 nm absorbance for each test and decreases
both minimum detection level and test range of the kit. 6.Cool all tubes to room temperature (RT). 7.Set the wavelength of spectrophotometer at OD 562 nm.
Calibrate the instrument to zero by using water. Subsequently,
measure the absorbance of all samples within 10 minutes. Note: Color development continues even after cooling to RT.
However, the subsequent development at RT is too weak to produce
significant error if all absorbance measurements are made within
10 minute. 8.Subtract OD562 of Blank from all readings. 9.Plot the BSA standard curve: OD562 (on Y axis) vs BSA
Standard concentration (on X axis). Use the standard curve to
determine the protein concentration of each unknown sample. . B. Microplate Procedure. 1. Mix BCA Reagent A and BCA Reagent B at 50:1. i.e., mix 50 ml
of BCA Reagent A with 1 ml BCA Reagent B. Note: Use the following formula to determine the total volume
of working reagent required.
(# standards+ # unknowns) x (# replicates) x (volume of working
reagent per sample)= total volume of working reagent required. 2.Follow Table 2 to prepare a fresh set of standards. (Dilute
Albumin (BSA) Standards with 0.9% NaCl or PBS). Table 2: Preparation of Albumin (BSA) Standards.
Tube Number Volume of
Diluent(ul) Volume of BSA (ul) Final BSA
Concentration (ug/ml)
A 0 ul 200 ul of 2 mg/ml
Stock 2000 ug/ml
B 30 ul 90 ul of tube A 1500
ug/ml
C 60 ul 60 ul of tube A
1000 ug/ml
D 60 ul 60 ul of tube B
750 ug/ml
E 60 ul 60 ul of tube C
500 ug/ml
F 60 ul 60 ul of tube E
250 ug/ml
G 60 ul 60 ul of tube F
125 ug/ml
H 100 ul 25 ul of tube G
25 ug/ml
I 60 ul 0 0 (blank)
. 3. Add 25 ul of each standard and protein samples into
separate microplate wells. 4. Add 200 ul of BCA working reagent to each well and mix
well. 5. Seal plates and incubate at 37°C for 30 minutes. 6. Cool plate to room temperature (RT). 7.Measure the absorbance at 562 nm on a plate reader within 10
minutes. 8. Subtract OD 562 of Blank from all readings.
Plot the BSA standard curve: OD562 (on Y axis) vs BSA Standard
concentration (on X axis). Use the standard curve to determine the
protein concentration of each unknown sample.
Troubleshooting
Problem Possible Cause Solution
No color in any tubes Sample contains a copper chelating
agent Dialyze, desalt or dilute sample Increase copper
concentration in working reagent (e.g., use 50:2, Reagent A:B)
Color of samples appears darker than
expected Protein concentration is too high. Sample
contains lipids or lipoproteins Dilute sample. Add 2%
SDS to the sample to eliminate interference from lipids3
Need to measure color at a different wavelength
Spectrophotometer or plate reader does not have 562nm
filter Color may be measure at any wavelength between
540nm and 590nm, although the slope of standard curve and overall
assay sensitivity will be reduced
规格 :
1 kit
宿主 :
No
储存 :
Upon receipt store at room temperature.
It is stable at room temperature for one year.
图像标记 :
Standard Curve of Net Absorbance versus protein sample concentration (Incubate at 37°C for 30 minutes).
更新 :
1/19/19 22:34
更多信息或购买 :
公司信息
武汉博士德生物工程有限公司
3942 B Valley Ave
Pleasanton, CA 94566
Pleasanton, CA 94566
boster@bosterbio.com
http://www.boster.com.cn925.485.4527
公司总部: 美国
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