BCA Protein Colorimetric Assay Kit

Catalog No: E-BC-K318-M

Method: Colorimetric method

Specification: 96T (Can detect 80 samples without duplication)

Instrument: Microplate reader

Sensitivity: 0.0165 mg/mL

Detection range: 0.0165-1 mg/mL

Please kindly provide us the lot number (on the outside of the box)
of the kit for more efficient service.
 General information

Intended use
This kit can be used to measure Total Protein (TP) content in serum, plasma,
culture cells, tissue and cells samples.

Background
The BCA protein concentration kit is an ideal protein quantification method
which is superior to the Lowry method. This method is fast and sensitive, stable
and reliable to different types of protein with small variation coefficient, which
is greatly favored by professionals. The BCA method is not affected by the
chemicals for most samples.

Detection principle
Cu2+ can be reduced to Cu+ by protein in alkaline condition. Cu+ can combine with
BCA reagent and form purple complex, which has a maximum absorption peak
at 562 nm. The absorbance value is proportional to the protein concentration.
Therefore, the protein concentration can be calculated according to the OD value.

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 Kit components & storage

Item Component Specification Storage

Reagent 1 BCA Reagent 25 mL × 1 vial RT, 12 months
Reagent 2 Copper Salt Solution 0.5 mL × 1 vial RT, 12 months

Reagent 3 Protein BSA Standard 1 mg × 1 vial RT, 12 months

Reagent 4 Standard Diluent 15 mL × 1 vial RT, 12 months

Microplate 96 wells No requirement
Plate Sealer 2 pieces

Note: The reagents must be stored strictly according to the preservation

conditions in the above table. The reagents in different kits cannot be mixed with
each other.

Materials prepared by users　

　　　　　 Instruments

Microplate reader (540-590 nm), Vortex mixer, Micropipettor, Incubator

　　　　　　 Consumptive material

Tips (10 μL, 200 μL, 1000 μL), EP tubes (1.5 mL, 2 mL, 5 mL)

　　　　　　 Reagents:

Double distilled water, Normal saline (0.9% NaCl), PBS (0.01 M, pH 7.4)

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！
Safety data
Some of the reagents in the kit contain dangerous substances. It should be
avoided to touch the skin and clothing. Wash immediately with plenty of water
if touching it carelessly. All the samples and waste material should be treated
according to the relevant rules of laboratory’s biosafety.

Precautions
！

Before the experiment, please read the instructions carefully, and wear gloves
and work clothes.

The key points of the assay

1. The time of incubation should be accurately.
2. The concentration of the sample protein must be diluted to 1 mg/mL or
less with normal saline, and it will show a good linear range below this
concentration.
3. Prevent the formulation of bubbles when adding the reagents to the
microplate.

Pre-assay preparation

Reagent preparation

1. Preparation of BCA working solution

Mix the reagent 1 and reagent 2 fully at a ratio of 50:1. Prepare the needed amount
solution before use. The prepared working solution can be stored at 4℃ for 24 h.
2. Preparation of 1 mg/mL standard solution
Dissolve a vial of reagent 3 powder with 1 mL reagent 4 and mix fully before
use. It is recommended to aliquot the prepared solution and it can be store at
-20℃ for 3 months.

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 Sample preparation
The samples should be prepared as conventional methods. Also please refer to
appendix II.
Sample requirements
The sample should not contain chelating agents (EGTA, EDTA) and reductive
substances (DTT, 2-mercaptoethanol).

Dilution of sample
It is recommended to take 2~3 samples with expected large difference to do
pre-experiment before formal experiment and dilute the sample according to
the result of the pre-experiment and the detection range (0.0165-1 mg/mL).

The recommended dilution factor for different samples is as follows (for

reference only)

Sample type Dilution factor

10% Mouse brain tissue homogenization 8-12

10% Mouse kidney tissue homogenization 8-12
Human serum 100-200
10% Rat liver tissue homogenization 15-20
10% Mouse heart tissue homogenization 8-12

Rat serum 100-200

Note: The diluent is normal saline (0.9% NaCl) or PBS (0.01 M, pH 7.4).

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 Assay protocol
Ambient temperature 25-30℃

Optimum detection wavelength 562 nm

Instructions for the use of transferpettor:

(1) Equilibrate the pipette tip in that reagent before pipetting each reagent.
(2) Don’t add the liquid outside the tips into the reaction system when pipetting
each reagent.

Plate set up

1 2 3 4 5 6 7 8 9 10 11 12

A A A S1 S9 S17 S25 S33 S41 S49 S57 S65 S73

B B B S2 S10 S18 S26 S34 S42 S50 S58 S66 S74

C C C S3 S11 S19 S27 S35 S43 S51 S59 S67 S75

D D D S4 S12 S20 S28 S36 S44 S52 S60 S68 S76

E E E S5 S13 S21 S29 S37 S45 S53 S61 S69 S77

F F F S6 S14 S22 S30 S38 S46 S54 S62 S70 S78

G G G S7 S15 S23 S31 S39 S47 S55 S63 S71 S79

H H H S8 S16 S24 S32 S40 S48 S56 S64 S72 S80

Note: A, blank wells; B-H, standard wells; S1-S80, sample wells.

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 Operating steps
The preparation of standard curve
Dilute 1 mg/mL BSA standard solution with normal saline to a serial
concentration. The recommended dilution gradient is as follows: 0, 0.2, 0.3, 0.4,
0.6, 0.7, 0.9, 1 mg/mL.

The measurement of samples

(1) Standard well: add 20 μL of standard solution with different concentration.
Sample well: add 20 μL of tested samples.
(2) Add 200 μL of BCA working solution to the wells of Step 1.
(3) Oscillate for 20 s to mix fully and incubate at 37℃ for 30 min.
(4) Measure the OD value of each well at 562 nm with microplate reader.

Operation table

Standard well Sample well

Standard solution with different concentration (μL) 20

Samples (μL) 20
BCA working solution (μL) 200 200

Oscillate for 20 s to mix fully and incubate at 37℃ for 30 min. Measure the
OD values of each well at 562 nm with microplate reader.

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 Calculation
Plot the standard curve by using OD value of standard and correspondent
concentration as y-axis and x-axis respectively. Create the standard curve with
graph software (or EXCEL). The concentration of the sample can be calculated
according to the formula based on the OD value of sample.

The standard curve is: y= ax + b.

Protein content (mg/mL) = (ΔA562 - b) ÷ a × f

Note:
y: The absolute OD value of standard
x: The concentration of Standard
a: The slope of standard curve
b: The intercept of standard curve
ΔA562: ODSample – ODBlank
f: Dilution factor of sample before test.

Notes
1. This kit is for research use only.
2. Instructions should be followed strictly, changes of operation may result in
unreliable results.
3. The validity of kit is 12 months.
4. Do not use components from different batches of kit.

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 Appendix I Performance characteristics

Appendix I Performance characteristics

Detection range 0.0165-1 mg/mL Average intra-assay CV (%) 2.2

Sensitivity 0.0165 mg/mL Average inter-assay CV (%) 4.5

Average recovery rate (%) 100

Example analysis
Dilute human serum with PBS (0.01 M,pH 7.4) for 50 times, take 0.02 mL of diluted
human serum and carry the assay according to the operation table.
The results are as follows:
standard curve: y = 0.88923x +0.03739, the average OD value of the sample well is
1.100, the average OD value of the blank well is 0.087, the calculation result is:
Protein content (mg/mL) =(1.100-0.087-0.03739) ÷ 0.88923 × 50 = 54.88 mg/mL

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 Appendix II Sample preparation

The following sample pretreatment methods are for reference only.

Serum
Collect fresh blood and stand at 25℃ for 30 min to clot the blood. Then centrifuge
at 2000 g for 15 min at 4℃ . Take the serum (which is the upper light yellow
clarified liquid layer) to preserve it on ice for detection. If not detected on the same
day, the serum can be stored at -80℃ for a month.

Plasma
Take fresh blood into the tube which has anticoagulant (Heparin is
recommended), centrifuge at 700-1000 g for 10 min at 4℃ . Take the plasma
(which is the upper light yellow clarified liquid layer, don't take white blood
cells and platelets in the middle layer) to preserve it on ice for detection. If not
detected on the same day, the plasma can be stored at -80℃ for a month.

Tissue sample
Take 0.02-1g fresh tissue to wash with homogenization medium at 2-8℃ . Absorb
the water with filter paper and weigh. Homogenize at the ratio of the volume
of homogenized medium (2-8℃ ) (mL): the weight of the tissue (g) =9:1, then
centrifuge the tissue homogenate for 10 min at 10000 g at 4℃ . Take the
supernatant to preserve it on ice for detection. If not detected on the same day,
the tissue sample (without homogenization) can be stored at -80℃ for a month.

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 Cells
Collect the cells and wash the cells with homogenization medium for 1~2
times. Centrifuge at 1000 g for 10 min and then discard the supernatant and
keep the cell sediment. Add homogenization medium at a ratio of cell number
(106): homogenization medium (μL) =1: 300-500. Sonicate or grind with hand-
operated in ice water bath. Centrifuge at 10000 g for 10 min, then take the
supernatant and preserve it on ice for detection. If not detected on the same
day, the cells sample (without homogenization) can be stored at -80℃ for a
month.

Note:
1. Homogenized medium: PBS (0.01 M, pH 7.4) or normal saline
2. Homogenized method:
(1) Hand-operated: Weigh the tissue and mince to small pieces (1 mm3),
then put the tissues pieces to glass homogenized tube. Add homogenized
medium into homogenized tube, place the tube into the ice bath with left
hand, and insert the glass tamping rod vertically into the homogenized tube
with the right hand to grind up and down for 6-8 min.
Or put the tissue into the mortar, and add liquid nitrogen to grind fully. Then
add the homogenized medium to homogenize.
(2) Mechanical homogenate: Weigh the tissue to EP tube, add the
homogenized medium to homogenize the tissue with homogenizer
instrument (60 Hz, 90s) in the ice bath. (For samples of skin, muscle and
plant tissue, the time of homogenization can be properly prolonged.)
(3) Ultrasonication: Treat the cells with ultrasonic cell disruptor (200 W, 2 s/
time, interval for 3 s, the total time is 5 min).

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 Notes for sample

1. Please predict the concentration before assaying. If the sample concentration

is not within the range of the standard curve, users must determine the
optimal sample dilutions for their particular experiments.
2. If the sample type is not included in the manual, a preliminary experiment is
suggested to verify the validity.
3. If a lysis buffer is used to prepare tissue homogenates or cell culture
supernatant, there is a possibility of causing a deviation due to the
introduced chemical substance.

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