Preparing Samples for Western Blot Analysis:

Protein Quantification
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After lysis of cells, it is important to determine the total protein concentration of the

sample. Accurate quantitation of the sample will allow you to load the proper amount of

protein in each lane. This avoids overloading the lane but still allows adequate detection

of the protein of interest. Proper quantitation is also critical when performing semi-

quantitative or quantitative Westerns. For accurate determine of relative amounts of

protein expression, the same amount of protein must be loaded into each lane.

There are several different methods for quantifying protein concentration in samples.

Some (e.g. measurement of nitrogen content or radioactively labeling cells) do not rely

upon absorptive properties of the protein sample. While these can be sensitive and

accurate assays, the more commonly used assays rely on spectrophotometric

determination of protein concentration and these will be the focus of this guide.

Spectrophotometric methods for measuring protein concentration are popular assays.

They are generally easy to ease, sensitive and do not rely on the use of hazardous

agents. There are several different methods and kits available for employing a

spectrophotometer to determine protein concentration. Choice of the best method

relies on various factors. A protein quantification assay should be easy to use and not be
cost prohibitive. The range of the assay should allow you to accurately quantify all

protein samples. Generally a linear response over a broad range is desired. The

sensitivity of the assay will determine the lowest level of protein that can be detected.

Assays also have different specificities; buffer components, such as detergents, can

interfere with the assay.

The different capabilities and limitations of several assays are discussed below.

Absorbance at 280 nm

A quick, but less specific measure of protein concentration is the absorbance of the

sample at 280 nm. Most proteins have an absorbance maxima at 280 nm as opposed to

nucleic acids which absorb at 260 nm. Aromatic residues, such as tryptophan, tyrosine

and phenylalanine are responsible for the observed absorbance, thus proteins lacking

these residues cannot be measured using this assay. Requires approximately 50 μg for

accurate readings.

Although nucleic acid contamination can interfere with measuring protein concentration

at 280 nm, a more accurate measure can be taken by measuring the absorbance of the

sample at both 260 nm and 280 nm and using the following calculation:

Protein mg/mL = 1.55 A280 – 0.76 A260

Advantages:

 Good for samples containing a single protein of known molar absorptivity

 Useful in chromotography to determine elution profile of protein

 Easy, quick assay

 Moderate sensitivity
Disadvantages:

 Need spectrophotometer capable of reading in the UV region

 Need to use quartz cuvettes

 Cannot use with proteins that do not contain aromatic residues

 Nucleic acid contamination can be a problem

Biuret Method

The biuret test is a test for detecting the presence of peptide bonds. Under alkaline

conditions, Cu2+ forms a violet-colored complex. Protein concentration is directly

proportional to the intensity of color, absorbed at 540 nm.

The Biuret reagent contains sodium hydroxide, hydrated copper (II) sulfate and

potassium sodium tartrate (to stabilize the complexes). Reduction of copper results in a

color change, which can be read at 550 nm. The linear range is typically 0.5-20mg

protein.

Advantages:

 Doesn’t rely on the amino acid composition of the protein

 Does not require a spectrophotometer capable of measuring in the UV region

 Good for whole tissue samples and other sources of high protein concentration

 Relatively few materials interfere with the assay

Disadvantages:

 Buffers, such as Tris and ammonia, can interfere with the reaction

 Cannot measure the concentration of proteins precipitated using ammonium sulfate

 Not as sensitive as other methods – requires higher amounts of protein

 Need to set up a standard curve

 Nucleic acid contamination can be a problem

 Proteins with abnormally high or low percentage of aromatic amino acids will give

high or low readings

The Lowry Method

The development of the Lowry method (named after Oliver Lowry) introduced a more

sensitive assay for determining protein concentration. The Lowry method is sensitive,

highly reproducible, inexpensive and easy to perform.

A modification of the Biuret test, the Lowry method relies on the reaction of copper

with proteins, but the sample is also incubated with the Folin-Ciocalteu reagent.

Reduction of the Folin-Ciocalteu reagent under alkaline conditions results in an intense

blue color (heteropolymolybdenum blue) that absorbs at 750 nm. The Lowry method is

best used with protein concentrations of 0.01–1.0 mg/mL.

Advantages:

 Easy to use

 Highly reproducible

 Inexpensive

 Sensitive and broad linear range

Disadvantages:

 Need to do a standard curve for every assay

 Timing and mixing of reagents must be precise

 Sensitivity depends on composition of protein as reaction partly dependent on polar

amino acids

 Interference from some buffers, particularly detergents

 Best suited for determining concentration of samples of the same protein rather

than as an absolute measurement

 Assay can be lengthy

Bicinchoninic Acid Assay (BCA)

BCA is similar to Lowry, except bicinchoninic acid (BCA) is used instead of the Folin-

Ciocalteu reagent. After reduction of Cu2+ ions, two molecules of BCA chelate with each

Cu+ ion resulting in formation of an intense purple color that absorbs at 560 nm.

BCA is as sensitive as the Lowry method and works well with protein concentrations

from 0.5 μg/mL to 1.5 mg/mL. Although detergents do not interfere as strongly as in

the Lowry method, other contaminants can interfere with the reaction. In addition,

aromatic amino acids can influence the reaction. However, at higher temperatures (37-

60°C), peptide bonds can also contribute to formation of the product. Therefore it is

recommended to perform the reaction at higher temperatures to increase the

sensitivity and decrease the variability due to amino acid composition.

Advantages:

 Easy to use

 Highly reproducible

 Inexpensive

 Sensitive and broad linear range

Disadvantages:

 Need to do a standard curve for every assay

 Interference from carbohydrates, catecholamines, tryptophan, lipids, phenol red,

cysteine, tyrosine, impure sucrose or glycerol, uric acid, iron and hydrogen peroxide
 Color continues to develop over time, but is stable for measurement after 30

minutes at 37°C

Dye-Binding Assays

Dye binding assays rely on an absorbance shift that occurs when a dye binds to proteins.

Several different dyes can be used: Coomassie Brillant Blue G-250, bromocresol green,

pyrogallol red and eosine y. The most commonly used dye-binding assay is the Bradford

Assay.

Bradford Assay

The Bradford assay, named after it’s developed, Marion M. Bradford, is an easy,

sensitive and accurate method for protein quantification. Binding of Coomassie Brillant

Blue G-250 to proteins, causes a shift of the dye from red (465 nm) to blue (595 nm)

under acidic conditions. It is compatible with more common reagents, although

detergents can cause interference. Proteins with a concentration of 20-2000 μg/mL can

be measured using the Bradford assay.

Advantages:

 Easy to use

 Sensitive and broad linear range

 Quick

 Compatible with many buffers

Disadvantages:

 Need to do a standard curve for every assay

 Reagent stains cuvettes

 Often need to dilute samples prior to analysis

 Depends strongly on amino acid composition

 Sensitive to detergents although some companies offer detergent-compatible

Bradford reagents