Protein Quantification

Biuret Principle

Cu²⁺ ions form a violet complex with peptide bonds (Cu²⁺ → Cu⁺)

• Abs @ 540nm

How long does Biuret take?

30 minutes

Biuret Linear Range

1-10 mg/ml

Biuret Interference

Limited to Soluble Proteins and Low sensitivity

Biuret Chemicals

Copper (II) Sulfate (CuSO4)

NaOH

K-Na-tartrate

Lowry Bronsted Principle

biuret reaction + reduction of Folin-Ciocalteau Reagent by tyrosine and tryptophan

• Abs @ 750nm

How long does Lowry take?

30 min to 1 hour

Linear Range for Lowry 

0‒200 μg/mL

Lowry Interference

Susceptible to interference (buffers, detergents, phenols)

Lowry Chemicals

Copper sulfate

Sodium hydroxide,

sodium or potassium tartrat

Folin Ciocalteau Reagent

BCA Principle

Cu²⁺ reduced to Cu⁺ → forms purple complex with BCA reagent

• Abs @ 562 nm

How long does BCA take?

30 minutes

BCA Linear Range

Standard - 0.01-2.5 mg/mL

Micro Assay - 0.4-100 μg/mL

BCA Advantage

Detergent compatible 

BCA Disadvantage

• Reaction strongly influenced by cysteine, tyrosine, and tryptophan

• Limited to Soluble Proteins

BCA Chemcials

Bichionic Acid

Copper  II Sulfate

Bradford Principle

Max. abs. of Coomassie Brilliant Blue G-250 dye changes from 465 nm to 595 nm after binding to proteins

How long does Bradford take? 

5 minutes

Bradford Interference

Non-linear standard curve, sensitive to detergents and buffer composition

Bradford Chemicals

Coomassie Brilliant Blue G-250 dye

Phosphoric acid and Methanol

UV Principle

Tryptophan and tyrosine absorb strongly at 280 nm.

can be determined using Beer’s Law with appropriate standard curves.

How long does UV take?

Immediate/None

UV Linear Range

0.1 to 1 AU or Protein Specific

UV Limitations

Only suitable for purified protein solutions

UV Chemicals

Tryptophan (Trp): 5500 M-1 cm-1

Tyrosine (Tyr): 1490 M-1 cm-1

Cystine: 120 M-1 cm-1

Ninhydrin Principle 

Amino Acids, Ammonia, and Primary Amino Groups in Proteins, when boiled in a pH 5.5 buffer with ninhydrin, form a purple color (abs @ 570 nm)

How long does Ninhydrin take?

24 hours

Ninhydrin Linear Range

1.0 to 50 mg/L

Ninhydrin Compatability

It reacts with primary and secondary amine groups

Ninhydrin Interference 

primary amines, sugars, reducing and oxidizing agents, and other amino acids

Ninhydrin Chemicals

acetone, ethanol, methanol, dimethyl sulfoxide (DMSO), or methyl cellosolve

Kjeldahl Principle

Total protein by nitrogen determination

How long does Kjeldahl take?

a few hours

Kjeldahl linear range 

0.1–20 mg/L

Kjeldahl Compatability

nearly all organic matrices containing easily convertible nitrogen

Kjeldahl Interference

Nitrate

Kjeldahl Chemicals

concentrated sulfuric acid, potassium sulfate, sodium hydroxide

Combustion Principle 

Measures the nitrogen gas produced from the complete

combustion (heat) of the sample

How long does combustion take?

A few minutes

Combustion linear range

1-2mg/ml or 200-300 mg

Combustion compatibility

organic nitrogen-containing samples

Combustion Interference

non-protein nitrogen

Combustion Chemicals 

chlorine trifluoride, nitric acid, and vanadium

Amino Acid Analysis Principle

breaks down protein into constituent amino acids. Then, separating amino acids

How long does Amino Acid Analysis take?

2-3 days, possible weeks

Amino Acid Linear Range

1 nmol - 100 nmol

Amino acid Analysis compatibility 

reagents, HPLC/UHPLC

Amino Acid Analysis Interference

amino acids, proteins, lipids, detergents, glycerol, and sugars

Amino acid analysis chemcials

Hydrochloric acid, Ninhydrin, Sodium citrate, lithium citrate

Biuret Assay Color

Purple

Lowry Color

Blue

Biuret Advantage 

quick, low interference from AA

Lowry Advantage

High sensitivity; measures aromatic AA

BCA color

Purple

Bradford Dye-Binding

Reddish (free dye)

Blue (protein bound)

Bradford Advantage 

Very fast, simple, inexpensive

UV Advantage

No reagents required; non-destructive

Ninhydrin Color

Purple

Tips for Choosing a Method: High sensitivity needed?

Use Lowry or BCA

Tips for Choosing a Method: Fast and simple?

Use Bradford

Tips for Choosing a Method: Detergents present?

 Use BCA (Bradford is sensitive to detergents)

Tips for Choosing a Method: Total nitrogen desired?

 Use Kjeldahl or Combustion

Tips for Choosing a Method: Pure protein sample?

UV Absorption works best