B6916
Synonym(s):
Coomassie dye binding protein assay, Coomassie dye binding protein assay, Protein dye reagent, Protein dye reagent
Warning
Eye Irrit. 2 - Met. Corr. 1 - Skin Irrit. 2 - STOT SE 2
Eyes,Central nervous system
8B - Non-combustible corrosive hazardous materials
WGK 1
Not applicable
Not applicable
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The field of proteomics is continually looking for new ways to investigate protein dynamics within complex biological samples. Recently, many researchers have begun to use RNA interference (RNAi) as a method of manipulating protein levels within their samples, but the ability to accurately determine these protein amounts remains a challenge. Fortunately, over the past decade, the field of proteomics has witnessed significant advances in the area of mass spectrometry. These advances, both in instrumentation and methodology, are providing researchers with sensitive assays for both identification and quantification of proteins within complex samples. This discussion will highlight some of these methodologies, namely the use of Multiple Reaction Monitoring (MRM) and Protein-AQUA.
To determine protein content, the Warburg-Christian method refers to measuring protein samples at 280 nm using a spectrophotometer.
Rules and good practice in sample preparation for Western blot sample preparation from cell culture and tissue samples.
Products for traditional and alternative protein quantitation techniques available, including BCA, Bradford, Lowry, and more.
Protein quantification methods, reagents, and immunoassay technology for accurately measuring the protein concentrations in a variety of samples.
For the Standard 3.1 mL Assay and 96 Well Plate Assay, the protein standards should be prepared in the range of 0.1 - 1.4 mg/mL. This is not the final concentration of the protein after being diluted in the assay.
For the full protocol, please refer to the document found here:
https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/165/479/b6916bul-ms.pdf
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Yes, phenol red at concentrations above 0.5 mg/mL will cause interference. Concentrations below 0.5 mg/mL are expected to be compatible.
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The color of this product can range from Faint Yellow-Brown to Light Brown. The exact result will be listed on the lot specific Certificate of Analysis. Please navigate to the ‘DOCUMENTATION’ section of the Product Detail Page to access a Certificate under ‘Certificate of Analysis’: https://www.sigmaaldrich.com/product/sigma/b6916#product-documentation
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A compatibility chart is listed in the bulletin. This chart indicates that this reagent is compatible with concentrations of SDS up to 0.125%.
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The Bradford method has a lower limit of detection of 20 ug/mL. The Lowry method has a lower limit of 10 ug/mL. Concentration of the sample may be necessary.
See the links below for additional helpful information:
Protein Quantitation Methods-
https://www.sigmaaldrich.com/applications/protein-biology/protein-quantitation
Amicon Centrifugal Filters-
https://www.sigmaaldrich.com/technical-documents/technical-article/protein-biology/protein-concentration-and-buffer-exchange/amicon-ultra-centrifugal-filters
Centricon Centrifugal Filters-
https://www.sigmaaldrich.com/substance/centriconplus70centrifugalfilter1234598765
Helpful?
Albumins are readily soluble in water and can only be precipitated by high concentrations of neutral salts such as ammonium sulfate. The solution stability of BSA is very good (especially if the solutions are stored as frozen aliquots). In fact, albumins are frequently used as stabilizers for other solubilized proteins (e.g., labile enzymes). However, albumin is readily coagulated by heat. When heated to 50°C or above, albumin quite rapidly forms hydrophobic aggregates which do not revert to monomers upon cooling. At somewhat lower temperatures aggregation is also expected to occur, but at relatively slower rates.
Please see the product data sheet which describes the solution stability of BSA:
https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/351/531/a8412pis.pdf
Helpful?
What is the useful concentration range that can be measured by Product B6916, Bradford Reagent?
The Bradford Reagent requires no dilution and is suitable for micro, multiwell plate, and standard (cuvet) assays. The linear concentration range is 0.1-1.4 mg/mL of protein, using BSA (bovine serum albumin) as the standard protein.
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When using Product B6916, Bradford Reagent, what can I do if I have a very dilute sample in a large volume?
The micro assay using this same reagent may be an option for you. The micro assay is used when a large volume (at least 1 mL) of a dilute sample is available for testing. The linear concentration range of this assay is lower than the standard or multiwell plate assays, (1-10 μg of total protein in 1 mL).
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Will the buffer or solution my protein is in interfere with Product B6916, Bradford Reagent?
A compatibility chart is listed in the bulletin. If your substance is not listed, then we recommend testing this by diluting the standard protein samples in the same buffer as the unknown samples.
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Is the BSA standard supplied with Product B6916, Bradford Reagent?
No, we do not supply the BSA standard with this reagent. We recommend using one of the following products: Protein Standard (BSA) Solution, (2 mg/mL), Product No. P0834 or Protein Standard (BSA) Solution, (1 mg/mL), Product No. P0914, if lower concentrations of protein are to be measured.
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