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The Pierce BCA Protein Assay – Reducing Agent Compatible

Working Range Characteristics/Advantages Applications Disadvantages Interfering Substances

Compatible with up to 5 mM Allows the use of the superior No microplate protocol is Compatible with all reducing
DTT, 35 mM 2-mercaptoethanol BCA Assay in situations in currently available agents and detergents found at
or 10 mM TCEP which it is normally unable to concentrations routinely used in
be read Requires heating for color protein sample buffers
No protein precipitation development
involved No precipitation step means
no worries about difficult-to-
Sample volume only 25 µl solubilize proteins

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 Applications Disadvantages Interfering Substances

Standard Protocol: Two stable reagents used to Adaptable for use with Not compatible with Reducing sugars and
20-2,000 g/ml make one working reagent microplates thiols/reducing agents reducing agents

Enhanced Working reagent stable for one Determine the amount of IgG Requires heating for Thiols
Standard Protocol: week at room temperature coated on plates color development
Copper chelating agents
5-250 g/ml Compatible with detergents Measure the amount of protein Not a true end-point assay
covalently bound to affinity Ascorbic acid and uric acid
Microplate Protocol: Simple, easy to perform supports3
20-2,000 g/ml Tyrosine, cysteine and
Less protein:protein variation Determine copper levels using a tryptophan
than Coomassie dye methods reagent formulated with BCA 50 mM Imidazole,
Works with peptides Reagent A 0.1 M Tris,
(three amino acids or larger) 1.0 M glycine
Flexible incubation protocols
allow customization of reagent
sensitivity and working range

The Micro BCA Protein Assay


Working Range Characteristics/Advantages Applications Disadvantages Interfering Substances

Standard Protocol: Three stable reagents used to Suitable for determining protein More substances interfere at Reducing sugars and
60˚C for 60 minutes make one working reagent concentration in very dilute lower concentrations than reducing agents
0.5-20 g/ml aqueous solutions with BCA Assay because the
Working reagent stable for sample volume-to-reagent Thiols
Microplate Protocol: 24 hours at room temperature Adaptable for use with volume ratio is 1:1
microplates Copper chelating agents
37˚C for 120 minutes Compatible with most detergents
1-20 g/ml 60˚C water bath is needed Ascorbic acid and uric acid
Simple, easy to perform
Tyrosine, cysteine and
Less protein:protein variation tryptophan
than BCA, Coomassie dye or
Lowry Methods 50 mM Imidazole,
0.1 M Tris,
Works with peptides 1.0 M glycine
(three amino acids or larger)
Linear color response to
increasing protein concentration
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