Revision for “Western Blotting: Protein Quantification” created on November 20, 2015 @ 19:28:28

Western Blotting: Protein Quantification
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. <strong class="dec" style="font-size: 18px; color: #9f2014;">Absorbance at 280 nm</strong> 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 <strong class="alpha" style="font-size: 16px; color: #444444;">Advantages:</strong> <ul> <li>Good for samples containing a single protein of known molar absorptivity</li> <li>Useful in chromotography to determine elution profile of protein</li> <li>Easy, quick assay</li> <li>Moderate sensitivity</li> </ul> <strong class="alpha" style="font-size: 16px; color: #444444;">Disadvantages:</strong> <ul> <li>Need spectrophotometer capable of reading in the UV region</li> <li>Need to use quartz cuvettes</li> <li>Can not use with proteins that do not contain aromatic residues</li> <li>Nucleic acid contamination can be a problem</li> </ul> <strong class="dec" style="font-size: 18px; color: #9f2014;">Biuret Method</strong> 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. <strong class="alpha" style="font-size: 16px; color: #444444;">Advantages:</strong> <ul> <li>Doesn’t rely on the amino acid composition of the protein</li> <li>Does not require a spectrophotometer capable of measuring in the UV region</li> <li>Good for whole tissue samples and other sources of high protein concentration</li> <li>Relatively few materials interfere with the assay</li> </ul> <strong class="alpha" style="font-size: 16px; color: #444444;">Disadvantages:</strong> <ul> <li>Buffers, such as Tris and ammonia, can interfere with the reaction</li> <li>Cannot measure the concentration of proteins precipitated using ammonium sulfate</li> <li>Not as sensitive as other methods – requires higher amounts of protein</li> <li>Need to set up a standard curve</li> <li>Nucleic acid contamination can be a problem</li> <li>Proteins with abnormally high or low percentage of aromatic amino acids will give high or low readings</li> </ul> <strong class="dec" style="font-size: 18px; color: #9f2014;">The Lowry Method</strong> 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. <strong class="alpha" style="font-size: 16px; color: #444444;">Advantages:</strong> <ul> <li>Easy to use</li> <li>Highly reproducible</li> <li>Inexpensive</li> <li>Sensitive and broad linear range</li> </ul> <strong class="alpha" style="font-size: 16px; color: #444444;">Disadvantages:</strong> <ul> <li>Need to do a standard curve for every assay</li> <li>Timing and mixing of reagents must be precise</li> <li>Sensitivity depends on composition of protein as reaction partly dependent on polar amino acids</li> <li>Interference from some buffers, particularly detergents</li> <li>Best suited for determining concentration of samples of the same protein rather than as an absolute measurement</li> <li>Assay can be lengthy</li> </ul> <strong class="dec" style="font-size: 18px; color: #9f2014;">Bicinchoninic Acid Assay (BCA)</strong> 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. <strong class="alpha" style="font-size: 16px; color: #444444;">Advantages:</strong> <ul> <li>Easy to use</li> <li>Highly reproducible</li> <li>Inexpensive</li> <li>Sensitive and broad linear range</li> </ul> <strong class="alpha" style="font-size: 16px; color: #444444;">Disadvantages:</strong> <ul> <li>Need to do a standard curve for every assay</li> <li>Interference from carbohydrates, catecholamines, tryptophan, lipids, phenol red, cysteine, tyrosine, impure sucrose or glycerol, uric acid, iron and hydrogen peroxide</li> <li>Color continues to develop over time, but is stable for measurement after 30 minutes at 37°C</li> </ul> <strong class="dec" style="font-size: 18px; color: #9f2014;">Dye-Binding Assays</strong> 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. <strong class="alpha" style="font-size: 16px; color: #444444;">Bradford Assay</strong> The Bradford assay, named after its 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. <strong class="roman" style="font-size: 14px; color: #444444;">Advantages:</strong> <ul> <li>Easy to use</li> <li>Sensitive and broad linear range</li> <li>Quick</li> <li>Compatible with many buffers</li> </ul> <strong class="roman" style="font-size: 14px; color: #444444;">Disadvantages:</strong> <ul> <li>Need to do a standard curve for every assay</li> <li>Reagent stains cuvettes</li> <li>Often need to dilute samples prior to analysis</li> <li>Depends strongly on amino acid composition</li> <li>Sensitive to detergents although some companies offer detergent-compatible Bradford reagents</li> </ul> Photo courtesy of <a href="" target="_blank">Will Jackson.</a>

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