The Pierce BCA Protein Assay – Reducing Agent Compatible
Working Range Characteristics/Advantages
Compatible with up to 5 mM
DTT, 35 mM 2-mercaptoethanol
or 10 mM TCEP
No protein precipitation
involved
Sample volume only 25 µl
Compatible with most
detergents
Significantly less (14–23%)
protein:protein variation than
Bradford-based methods
Colorimetric method: measure
The BCA Protein Assay
Working Range Characteristics/Advantages
Standard Protocol: Two stable reagents used to
20-2,000 g/ml make one working reagent
Enhanced Working reagent stable for one
Standard Protocol: week at room temperature
5-250 g/ml Compatible with detergents
Microplate Protocol: Simple, easy to perform
20-2,000 g/ml
Less protein:protein variation
than Coomassie dye methods
Works with peptides
(three amino acids or larger)
Flexible incubation protocols
allow customization of reagent
sensitivity and working range
The Micro BCA Protein Assay
Working Range Characteristics/Advantages
Standard Protocol: Three stable reagents used to
60˚C for 60 minutes make one working reagent
0.5-20 g/ml
Working reagent stable for
Microplate Protocol: 24 hours at room temperature
37˚C for 120 minutes Compatible with most detergents
1-20 g/ml
Simple, easy to perform
Less protein:protein variation
than BCA, Coomassie dye or
Lowry Methods
Works with peptides
(three amino acids or larger)
Linear color response to
increasing protein concentration
Applications
Allows the use of the superior
BCA Assay in situations in
which it is normally unable to
be read
No precipitation step means
no worries about difficult-to-
solubilize proteins
Applications
Adaptable for use with
microplates
Determine the amount of IgG
coated on plates
Measure the amount of protein
covalently bound to affinity
supports3
Determine copper levels using a
reagent formulated with BCA
Reagent A
Applications
Suitable for determining protein
concentration in very dilute
aqueous solutions
Adaptable for use with
microplates
Disadvantages
No microplate protocol is
currently available
Requires heating for color
development
Disadvantages
Not compatible with
thiols/reducing agents
Requires heating for
color development
Not a true end-point assay
Disadvantages
More substances interfere at
lower concentrations than
with BCA Assay because the
sample volume-to-reagent
volume ratio is 1:1
60˚C water bath is needed
Interfering Substances
Compatible with all reducing
agents and detergents found at
concentrations routinely used in
protein sample buffers
Interfering Substances
Reducing sugars and
reducing agents
Thiols
Copper chelating agents
Ascorbic acid and uric acid
Tyrosine, cysteine and
tryptophan
50 mM Imidazole,
0.1 M Tris,
1.0 M glycine
Interfering Substances
Reducing sugars and
reducing agents
Thiols
Copper chelating agents
Ascorbic acid and uric acid
Tyrosine, cysteine and
tryptophan
50 mM Imidazole,
0.1 M Tris,
1.0 M glycine
The Modified Lowry Protein Assay
Working Range Characteristics/Advantages Applications Disadvantages Interfering Substances

Standard Protocol: Two-reagent system— Lowry method is the most cited Timed addition of Folin reagent Detergents (cause
1-1,500 g/ml shelf life of at least one year protein assay in the literature adds complexity precipitation)

Two-step incubation requires precise Adaptable for use with Longer total assay time Thiols, disulfides
sequential timing of samples microplates
Practical limit of about Copper chelating reagents
Color response read at 750 nm 20 samples per run
Carbohydrates including
Works with peptides (three amino acids hexoseamines and their
or larger) N-actyl derivatives

Protein:protein variation similar to that Glycerol, Tris, Tricine, K+1 ions

seen with BCA Method
Many authors have reported ways to deal
with substances that interfere

Coomassie Plus – The Better Bradford™ Assay

Working Range Characteristics/Advantages Applications Disadvantages Interfering Substances

Linear Range: Simple/fast protocols Standard assay Less linear color response Detergents
IgG: 125-1,500 g/ml in the micro assay
Total preparation and assay time Micro assay
BSA: 125-1,000 g/ml <30 minutes Effect of interfering substances
Microplate format assay
more pronounced in the
Standard Assay: One reagent system; stable for 12 months Assay of protein solutions micro assay
Sample-to-Reagent Ready-to-use formulation — no dilution containing reducing agents
Ratio: 1:30 Protein dye complex has
or filtration needed Quantitation of
Typical Working Range: tendency to adhere to glass
100-1,500 g/ml Nearly immediate color development at immobilized protein (easily removed with MeOH)
room temperature Protein in permeabilized cells Protein must be >3,000 Da
Micro Assay: Linear color response in standard assay
Sample-to-Reagent NaCNBH3 determination
(more accurate results)
Ratio: 1:1
Typical Working Range: Color response sensitive to changes in pH
1-25 g/ml Temperature dependence of
color response
Compatible with buffer salts, metal ions,
reducing agents, chelating agents
Low-odor formulation

Coomassie (Bradford) Protein Assay

Working Range Characteristics/Advantages Applications Disadvantages Interfering Substances

Standard Assay: Simple-to-perform protocols Standard assay Nonlinear color response Detergents
Sample-to-Reagent
One-reagent system, stable for 12 months Micro assay More protein standard
Ratio: 1:50
concentrations required to cover
100-1,500 g/ml Ready-to-use formulation Microplate format assay
working range
No dilution or filtration needed Assay of protein solutions
Micro Assay: containing reducing agents Micro assay has potential for
Sample-to-Reagent Fast, nearly immediate color development interference
Ratio: 1:1 at room temperature Cell line lysates
Protein must be >3,000 Da
1-25 g/ml Total preparation and assay time Protein recovery studies
<30 minutes
Typical protein:protein variation expected for
a Coomassie dye-based reagent
Color response sensitive to pH
Temperature-dependent color response
Compatible with buffer salts, metal
ions, reducing agents, chelating agents