WSE-7520 EzBCA Protein Assay Kit

Purpose and Application

Protein quantification by BCA method

Features

Quantification of proteins extracted from cells, tissues, and microorganisms
Suitable for protein quantification of electrophoresis samples using the BCA method
Total protein assay kit based on the BCA method
Rapid, simple, and highly accurate detection
Reduced interference from reducing agents and chelating agents through pretreatment
Includes pretreatment reagents to minimize the effects of reducing agents
Supplied with standard proteins for calibration (BSA and BGG)
Low interference from coexisting substances, ensuring excellent linearity and high accuracy
Measurement at 562 nmImmediate detection after mixing with protein (measurement of absorbance at 595 nm)

Description

Principle of the BCA Protein Assay

The BCA (Bicinchoninic Acid) method is a protein quantification assay based on the formation of a complex between proteins (peptides consisting of three or more amino acids) and copper ions. Amino acids with reducing properties—such as Cys, Tyr, and Trp—as well as peptide bonds, reduce copper(II) ions (Cu²⁺) to copper(I) ions (Cu⁺) in solution. Under alkaline conditions, BCA reacts with Cu⁺ to form a purple-colored complex. The absorbance of this complex is measured at 562 nm, and the protein concentration is determined using a calibration curve generated with standard proteins.

Although the reaction is compatible with detergents, it may be affected by reducing agents, thiols, glucose, ammonium sulfate, and other interfering substances.
Reference: Smith P. K., Analytical Biochemistry 150, 76–85 (1985).

The WSE-7520 EzBCA Protein Assay Kit is designed for total protein quantification using the BCA method. The kit includes BCA reagents (A and B), standard proteins (BSA and BGG), and pretreatment reagents to minimize interference from reducing agents. The recommended quantification ranges are 15–2000 μg/mL for the standard assay and 3–100 μg/mL for the low-concentration assay.

Accurate quantification with good linearity even in the presence of reducing agents

A dilution series of BGG standard was prepared with a final concentration of 20 mM DTT (BGG_DTT), and protein quantification was performed using the EzBCA Protein Assay Kit. In the presence of 20 mM DTT, color development was inhibited, and accurate quantification was not possible.

However, when the DTT-containing BGG samples were pretreated (BGG_DTT_Pretreat), the inhibitory effect of DTT was reduced, enabling accurate quantification with good linearity comparable to that of untreated BGG without DTT.

In addition, no differences in color development or measured values were observed between pretreated and non-pretreated BGG standard samples without DTT, indicating that the pretreatment step does not affect the assay performance.

These results demonstrate that the EzBCA Protein Assay Kit effectively reduces interference from reducing agents, allowing reliable protein quantification.

High correlation between electrophoresis patterns and total protein quantification by the BCA method

The graphs below show the results of protein quantification using the EzBCA Protein Assay Kit for proteins extracted from HeLa cells using EzApply, EzRIPA Lysis Kit, EzProteoLysis Native, and EzBactYeast Crusher. After extraction, samples were incubated at 37°C for 10 minutes with either pretreatment reagent (Pretreatment) or distilled water (DW), followed by quantification.

The left graph shows the quantification results of samples immediately after extraction, while the right graph shows results for electrophoresis samples prepared by adding DTT to the extracts and heating at 95°C for 5 minutes.

In addition, the electrophoresis patterns of these proteins, separated using u-PAGEL (UH-T420), are shown alongside. The gel images were analyzed using a CS analyzer, and the integrated intensity values of each lane were calculated and compared with the quantification results. Each graph plots relative protein concentration against relative integrated intensity.

These results demonstrate that protein quantification using the EzBCA Protein Assay Kit with pretreatment shows a high correlation with electrophoresis-based analysis.

Comparison of Protein Assay Kits

	WSE-7510 EzBradford Protein Assay Kit	WSE-7520 EzBCA Protein Assay Kit
Features	• Suitable for protein/electrophoresis samples • Rapid, simple, accurate • Not affected by reducing agents or chelators • Includes Protein Diluent (reduces detergent effects) • Includes standards (BSA, BGG) • One-reagent system	• Suitable for protein/electrophoresis samples • Rapid, simple, accurate • Less affected by detergents • Includes pretreatment reagents (reduces reducing agent interference) • Includes standards (BSA, BGG) • Low interference, high linearity & accuracy
Reagent Preparation	Not required	Mix Reagent A:B = 50:1 before use
Standard Assay	Sample: 10 μL Reagent: 200 μL	Sample: 25 μL Reagent: 200 μL 37°C, 30 min
Low Range Assay	Sample: 50 μL Reagent: 150 μL	Sample: 25 μL Reagent: 200 μL 60°C, 30 min
Wavelength	595 nm	562 nm
Capacity	~2,500 wells
Contents	1. Bradford Reagent (500 mL) 2. Protein Diluent (50 mL) 3. BSA (2 mg/mL, 10 mL) 4. BGG (2 mg/mL, 10 mL)	1. BCA Reagent A (500 mL) 2. BCA Reagent B (12 mL) 3. Pretreatment Solution (50 mL) 4. Pretreatment Agent (800 mg) 5. BSA (2 mg/mL, 10 mL) 6. BGG (2 mg/mL, 10 mL)
Storage	Refrigerated, light-protected (1 year)

Permissible Concentrations of Coexisting Substances

The table below summarizes the permissible concentrations of coexisting substances in sample solutions that do not affect the reaction system of protein quantification reagents. Please note that, depending on the type and concentration of protein, the combination of reagents, and conditions such as pH, the reaction system may still be affected even at concentrations lower than those listed in the table.

	No additives	Surfactant-containing sample / Protein Diluent added
WSE-7420 EzRIPA Lysis kit	0.5x	0.5x
WSE-7424 EzProteoLysis Native	1x	1x
WSE-7423 EzBactYeast Crusher	0.5x	1x
AE-1430 EzApply	0.063x	1x
Sodium Deoxycholate	-	10%
SDS	-	0.63%
Triton X-100	0.3%	2.5%
NP-40	0.63%	2.5%
Tween 20	0.63%	0.63%
CHAPS	10%	10%
N-Octyl-β-D-glucopyranoside	1.3%	10%
n-Octyl-β-D-thioglucoside	2.5%	10%
Dodecyl-β-D-maltoside	1.25%	2.5%
2-Mercaptoehanol	40mM	40mM
Dithiothreitol	40mM	40mM
TCEP	40mM	40mM
Glutathione	-	-
Cysteine	40mM	40mM
Tris/pH8.0	1M	1M
Tricine/pH8.0	1M	1M
HEPES/pH8	500mM	1M
MOPS/pH8	1M	1M
Sodium Phosphate Buffer	500mM	1M
PBS	2x	2x
TBS	1x	1x
Ammonium sulfate	4M	4M
Urea	6M	3M
Thiourea	2M	2M
Trichloroacetic acid	750mM	750mM
N,N-dimethylformamide	20%	20%
Dimethyl sulfoxide	20%	20%
Glycerol	20%	20%
Glucose	20%	20%
Sucrose	20%	20%
CaCl2	100mM	100mM
MgCl2	125mM	250mM
NaOH	250mM	250mM
HCL	500mM	500mM
MeOH	40%	40%
EtOH	40%	40%
EDTA	200mM	200mM
EGTA	40mM	40mM

Instruction Manual

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Specifications

	WSE-7520 EzBCA Protein Assay Kit
Contents	① BCA Reagent A: 500 mL ② BCA Reagent B: 12 mL ③ Pretreatment Solution: 50 mL ④ Pretreatment Agent: 800 mg ⑤ BSA Standard (2 mg/mL Bovine Serum Albumin): 10 mL ⑥ BGG Standard (2 mg/mL Bovine Gamma Globulin): 10 mL
Capacity	Approx. 2,500 wells
Sample Volume	Working Reagent Mix BCA Reagent A and B at a ratio of 50:1 immediately before use. Standard Assay Protein sample: 25 µL/well Working reagent: 200 µL/well Incubate at 37°C for 30 minutes. *Low-Concentration Assay Protein sample: 25 µL/well Working reagent: 200 µL/well Incubate at 60°C for 30 minutes.
Storage	Store refrigerated, protected from light
Shelf Life	1 year (unopened, from date of manufacture)

Ordering Information

Code No.	Description	Unit
2332384	WSE-7520 EzBCA Protein Assay Kit	1 set