PROTEIN ASSAYS

Biochemistry of Proteins

Protein Assays - Introduction

Protein Assays introduction

The determination of proteins in solution is performed by various methods. Among these, colorimetric methods are widely used, because of interesting features especially conveniency, sensitivity.

Mesure of absorbance	Generally based on the optical absorbance of peptidic bounds (215 nm) or aromatic amino-acids (280 nm). Very simple and rapid. The user should know the exact extinction coefficient of the assayed protein(s). Furthermore, this methods is often leantover by, or does not suit samples with numerous compounds in buffer (Salts, DMSO, detergents…), nor UV absorbing contaminants (for example hemoglobins)
Kjendal method	Colorimetric assay; very low sensitivity
Biuret method	Colorimetric assay, based on the reduction of Cu++ by peptidic bond in alkalin conditions; low sensitivity
Lowry method	Improved Biuret assay (the folin-Ciocalteu reagent increases the color development); reading at 750 nm ; not very convenient (prepare freshly reagents each day; 2 incubations, timed operating and controler of temperature); noticable interferences with compounds found in biological samples
Bicinchoninic method	Colorimetric assay, based on the chelating of Cu++ ions by bicinchoninic acid producing an intense purple colour; very linear and wide standard curve.
Bradford method	Colorimetric assay based on the interaction of a dye (Coomassie) with some amino-acids (notably charged ones); well known and used, but a lot of modified procedures. Important protein-to-protein signal variations.

How to choose the right Protein Assay?

Every protein assay exhibits restrictions of use depending on applications. The most useful features to consider are :

Sensitivity (lower detection level)
Variation of signal from protein to protein
Compatibility with substances found in samples
Linearity of signal with protein concentration

Uptima offers 3 colorimetric assays that cover most applications. These high quality and optimized formulated reagents overcome most of conventionnal methods, whilst fulfiling most requirement of sensitivity, compatibility, accuracy and ease-of-use.

Select the most important features and find in the following table the assay that answers the best to your requirements, in regards of performance :

Product

Suits especially to applications where are needed

Drawbacks

BC Assay

UP40840

Accuracy, versatility, compatibility*, along with good sensitivity

(*notably with detergents, bases, nucleic acids, lipids…)

Incompatible with reducing agents, some chelators

BC Assay

MicroBC Assay

As BC Assay, but 4-6 fold more sensitive !

Same as BC Assay

MicroBC Assay

Coo Assay

Quick of use, and compatibility with reducing agents

Incompatible with most detergents, lipids, alkalis…

Protein to protein variations

Coo Assay

BSA protein standard	UP36859A	1x10ml
	UP36859D	30ml
Bovine serum albumin calibrated at 2 mg/ml, in water, for protein assays

BC Assay

UP40840A

MicroBC Assay

UP75860A

Coo Assay (Bradford)

UPF86400

(modified lowry assay, based on bicinchoninic acid)

(modified lowry assay, based on bicinchoninic acid)

(modified Bradford assay, based on Coomassie dye)

A very versatile and efficient assay, suitable for most applications. Detergents compatible.

The high sensitivity version of the BC Assay

A very popular assay, worldwide known and documented. Reducing agent compatible.

Protocol

Standard Assay

Room

Temperature

Enhanced

Standard Assay

Broad

range

Intermediate

High Sensitivity

Max Sensitiivity

Reagent

Mix 2 components A+B (50:1)

mix 3 components

A+B+C (25:25:1)

1 ready-to-usereagent

(Coo)

1 ready-to-use reagent

(Coo)

Sample (in tube microplate)

100µl

(25µl)

100µl

(25µl)

100µl

(25µl)

1ml (150µl)

40µl

(5µl)

50µl

(10µl)

200µl

(25µl)

1ml

(150µl)

Reagent

2 ml

(200µl)

2 ml

(200µl)

2 ml

(200µl)

1ml (150µl)

2ml

(250µl)

1.5ml

(300µl)

2ml

(250µl)

1ml

(150µl)

Incubation

30min at

37°C

2h at Room

Temperature

30mn at

60°C

1h at 37°C

1min at Room Temperature

OD reading

562nm (540-590nm)

595nm (570-610nn)

Comments

Flexible protocols (temperature and duration)

(One minute protocole with microwave heating)

Great accuracy

Very easy and quick

No filtration needed!

Corrosive

Performances / features Overcomes often Coomassie assay

Accuracy, Linearity

Very good, 20-1000µg/ml

Very good, 1-100µg/ml

50-1500µg/ml

50-800µg/ml

20-200µg/ml

1-25µg/ml

Protein to protein variations

Low (3-4 fold lower than with the Coo Assay)

Low (slightly better than BC Assay)

Noticable

Sensitivity and working range

20-2000µg/ml

5-500µg/ml

1-200µg/ml

50-2000µg/ml

50-1000µg/ml

20-500µg/ml

1-100µg/ml

Stability

>1 year at Room Temperature

1 year at 4°C

Compatibility

Most detergents, chaotropic agents, preservatives (anti-microbials…), inhibitors (of proteases…), buffer and salts

Similar to BC Assay, but compatible concentrations are slighly lower because of sample/reagent volume ratio

Reducing agents (80mM DTT…),

2% Triton X100, 400mM Bicarbonate…

Incompatibility

Reducing agents (sugars, thiols…), copper chelators, cys, tyr, try amino-acids

Same as for the BC Assay

Incompatible with numerous detergents, strong Alkaline solutions

Applications

Samples

Purified proteins, complex mixtures, polypeptides, immobilized proteins...

Notably containing: nucleic acids, lipids, detergents…

Alkalin samples

Purified proteins, complex mixtures, polypeptides...

Notably containing: nucleic acids, lipids, detergents…

Alkalin samples

Purified proteins, complex mixtures

Acidic samples

Reducing agents containing samples

Others

Determination of Copper concentration

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