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<p>This is the protocol to be used in the determination of total protein in brain tissue by the Pierce BCA method. BSA (Bovine Serum Albumin) is used as the standard and samples are prepared by sonification in 1% (w/v) SDS.</p>
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[GUIDELINES]
<p align="LEFT"><strong>INTRODUCTION</strong></p>
<p><span style="font-size: small;">The Thermo Scientific™ Pierce™ BCA Protein Assay is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein. This method combines the well-known reduction of Cu</span><sup><span style="font-size: xx-small;">+2 </span></sup><span style="font-size: small;">to Cu</span><sup><span style="font-size: xx-small;">+1 </span></sup><span style="font-size: small;">by protein in an alkaline medium (the biuret reaction) with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu</span><sup><span style="font-size: xx-small;">+1</span></sup><span style="font-size: small;">) using a unique reagent containing bicinchoninic acid. </span><span style="font-size: small;">The purple-colored reaction product of this assay is formed by the chelation of two molecules of BCA with one cuprous ion. This water-soluble complex exhibits a strong absorbance at 562nm that is nearly linear with increasing protein concentrations over a broad working range (20-2000μg/mL). The BCA method is not a true end-point method; that is, the final color continues to develop. However, following incubation, the rate of continued color development is sufficiently slow to allow large numbers of samples to be assayed together. </span></p>
<p><span style="font-size: small;">The macromolecular structure of protein, the number of peptide bonds and the presence of four particular amino acids (cysteine, cystine, tryptophan and tyrosine) are reported to be responsible for color formation with BCA. </span><span style="font-size: small;">Studies with di-, tri- and tetrapeptides suggest that the extent of color formation caused by more than the mere sum of individual color-producing functional groups. </span><span style="font-size: small;">Accordingly, protein concentrations generally are determined and reported with reference to standards of a common protein such as bovine serum albumin (BSA). A series of dilutions of known concentration are prepared from the protein and assayed alongside the unknown(s) before the concentration of each unknown is determined based on the standard curve. If precise quantitation of an unknown protein is required, it is advisable to select a protein</span></p>
<p>standard that is similar in quality to the unknown; for example, a bovine gamma globulin (BGG) standard may be used when assaying immunoglobulin samples.</p>
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<p><strong>TRAINING REQUIREMENTS</strong></p>
<p>Able to use a plate reader and to accurately dilute tissue samples.</p>
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<p><strong>REFERENCES</strong></p>
<p><u>BCA Protein Assay Reagent</u> (instruction manual), Pierce. Rockford, IL.</p>
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<p><u>Softmax User’s Manual</u>, Molecular Devices Corporation. Sunnyvale, CA.</p>
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