Name: linearization of the bradford protein assay
Uploaded: 2010-04-12T17:00:00
Description: Watch this Scientific Journal Video about Linearization of the Bradford Protein Assay at JoVE.com

<p class="jove_content">This paper is dedicated to the memory of the late Dr. Zvi Selinger, who hosted the original research described here.</p>

<p class="jove_title">Linearization of the Bradford Protein Calibration Graph:</p>
<p class="jove_content">The Coomassie brilliant blue protein assay, commonly known as the Bradford assay <sup>1</sup>, is widely used because of its rapid and convenient protocol as well as its relative sensitivity. Unfortunately, there is a large degree of curvature over a broad range of protein concentrations (Fig. 1). Therefore, only a narrow range of relatively high protein concentrations, 2-10 &#956;g/ml BSA, is used for the calibration graph, which then bet...

<ol>
	<li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> <em style='font-style: italic'>Anal Biochem</em>. <b>72</b>, 248-254 (1976).</li><li>Chial, H. J., Thompson, H. B., Splittgerber, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+spectral+study+of+the+charge+forms+of+Coomassie+blue+G.">A spectral study of the charge forms of Coomassie blue G.</a> <em style='font-style: italic'>Anal Biochem</em>. <b>209</b>, 258-266 (1993).</li><li>Chial, H. J., Splittgerber, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+the+binding+of+Coomassie+brilliant+blue+to+proteins+at+low+and+neutral+pH.">A comparison of the binding of Coomassie brilliant blue to proteins at low and neutral pH.</a> <em style='font-style: italic'>Anal Biochem</em>. <b>213</b>, 362-369 (1993).</li><li>Compton, S. J., Jones, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+dye+response+and+interference+in+the+Bradford+protein+assay.">Mechanism of dye response and interference in the Bradford protein assay.</a> <em style='font-style: italic'>Anal Biochem</em>. <b>151</b>, 369-374 (1985).</li><li>Congdon, R. W., Muth, G. W., Splittgerber, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+binding+interaction+of+Coomassie+blue+with+proteins.">The binding interaction of Coomassie blue with proteins.</a> <em style='font-style: italic'>Anal Biochem</em>. <b>213</b>, 407-413 (1993).</li><li>Zor, T., Selinger, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linearization+of+the+Bradford+protein+assay+increases+its+sensitivity:+theoretical+and+experimental+studies.">Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies.</a> <em style='font-style: italic'>Anal Biochem</em>. <b>236</b>, 302-308 (1996).</li></ol>

<p class="jove_content">The Bradford protein assay is popular due to its ease of performance and relative sensitivity. The linearization over the entire protein concentrations range obtained by the protocol presented here further simplifies the assay, as the unknown samples do not need to fall within the range of the calibration graph.</p>
<p class="jove_content">Importantly, the improved protocol further provides the following advantages over the original Bradford protocol:</p>
<ol>
  <li>Increased accuracy.</li>
  <li>The sensitivity is incre...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Bradford reagent</td>
<td>&nbsp;</td>
<td>Bio-Rad Laboratories</td>
<td>500-0006</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Serum Albumin</td>
<td>&nbsp;</td>
<td>Amersco</td>
<td>0332</td>
<td>&nbsp;</td>
<tr>
<td>Multiplate absorbance reader</td>
<td>&nbsp;</td>
<td>BioTek</td>
<td>Synergy2</td>
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linearization of the bradford protein assay

Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, the ratio of the absorbance measurements at 590 nm and 450 nm is strictly linear with protein concentration. This simple procedure increases the accuracy and improves the sensitivity of the assay about 10-fold, permitting quantification down to 50 ng of bovine serum albumin. Furthermore, the interference commonly introduced by detergents that are used to create the cell lysates is greatly reduced by the new protocol. A linear equation developed on the basis of mass action and Beer's law perfectly fits the experimental data.

Watch this Scientific Journal Video about Linearization of the Bradford Protein Assay at JoVE.com

Linearization of the Bradford Protein Assay

<p class="jove_content">The accuracy and sensitivity of protein determination by the rapid and convenient Bradford assay is compromised by intrinsic nonlinearity. We show a simple linearization procedure that greatly increases the accuracy, improves the sensitivity of the assay about 10-fold, and significantly reduces interference by detergents.</p>

Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. ...

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