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    Bradford Protein Assay

    Uploaded by

    Farhan Ahmad
    The Bradford assay is a fast and accurate method for determining protein concentration using Coomassie Brilliant Blue dye. The absorbance maximum of the dye shifts from 465nm to 595nm when bound to protein, allowing simple spectrophotometric quantification. The assay works over a 10-fold concentration range and is useful for determining protein content in cell fractions and assessing concentrations for gel electrophoresis. The procedure involves preparing protein standards, adding dye reagent to samples and standards, incubating, and measuring absorbance at 595nm to generate a standard curve and determine sample concentrations.

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    Bradford Protein Assay

    Uploaded by

    Farhan Ahmad
    200 views3 pages
    The Bradford assay is a fast and accurate method for determining protein concentration using Coomassie Brilliant Blue dye. The absorbance maximum of the dye shifts from 465nm to 595nm when bound to protein, allowing simple spectrophotometric quantification. The assay works over a 10-fold concentration range and is useful for determining protein content in cell fractions and assessing concentrations for gel electrophoresis. The procedure involves preparing protein standards, adding dye reagent to samples and standards, incubating, and measuring absorbance at 595nm to generate a standard curve and determine sample concentrations.

    Original Description:

    Various protein present in our body.This is the method to check protein.

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    The Bradford assay is a fast and accurate method for determining protein concentration using Coomassie Brilliant Blue dye. The absorbance maximum of the dye shifts from 465nm to 595nm when bound to protein, allowing simple spectrophotometric quantification. The assay works over a 10-fold concentration range and is useful for determining protein content in cell fractions and assessing concentrations for gel electrophoresis. The procedure involves preparing protein standards, adding dye reagent to samples and standards, incubating, and measuring absorbance at 595nm to generate a standard curve and determine sample concentrations.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    200 views3 pages

    Bradford Protein Assay

    Uploaded by

    Farhan Ahmad
    The Bradford assay is a fast and accurate method for determining protein concentration using Coomassie Brilliant Blue dye. The absorbance maximum of the dye shifts from 465nm to 595nm when bound to protein, allowing simple spectrophotometric quantification. The assay works over a 10-fold concentration range and is useful for determining protein content in cell fractions and assessing concentrations for gel electrophoresis. The procedure involves preparing protein standards, adding dye reagent to samples and standards, incubating, and measuring absorbance at 595nm to generate a standard curve and determine sample concentrations.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    of 3

    Bradford protein assay

    Considerations for use


    The Bradford assay is very fast and uses about the same amount of protein as the
    Lowry assay. It is fairly accurate and samples that are out of range can be retested
    within minutes. The Bradford is recommended for general use, especially for
    determining protein content of cell fractions and assesing protein concentrations for
    gel electrophoresis.
    Assay materials including color reagent, protein standard, and instruction booklet are
    available from Bio-Rad Corporation. The method described below is for a 100 l
    sample volume using 5 ml color reagent. It is sensitive to about 5 to 200 micrograms
    protein, depending on the dye quality. In assays using 5 ml color reagent prepared in
    lab, the sensitive range is closer to 5 to 100 g protein. Scale down the volume for the
    "microassay procedure," which uses 1 ml cuvettes. Protocols, including use of
    microtiter plates are described in the flyer that comes with the Bio-Rad kit.

    Principle
    The assay is based on the observation that the absorbance maximum for an acidic
    solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when
    binding to protein occurs. Both hydrophobic and ionic interactions stabilize the
    anionic form of the dye, causing a visible color change. The assay is useful since the
    extinction coefficient of a dye-albumin complex solution is constant over a 10-fold
    concentration range.

    Equipment
    In addition to standard liquid handling supplies a visible light spectrophotometer is
    needed, with maximum transmission in the region of 595 nm, on the border of the
    visible spectrum (no special lamp or filter usually needed). Glass or polystyrene
    (cheap) cuvettes may be used, however the color reagent stains both. Disposable
    cuvettes are recommended.

    Procedure
    Reagents

    1. Bradford reagent: Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml


    95% ethanol, add 100 ml 85% (w/v) phosphoric acid. Dilute to 1 liter when the
    dye has completely dissolved, and filter through Whatman #1 paper just before
    use.
    2. (Optional) 1 M NaOH (to be used if samples are not readily soluble in the color
    reagent).
    The Bradford reagent should be a light brown in color. Filtration may have to be
    repeated to rid the reagent of blue components. The Bio-Rad concentrate is expensive,
    but the lots of dye used have apparently been screened for maximum effectiveness.
    "Homemade" reagent works quite well but is usually not as sensitive as the Bio-Rad
    product.
    Assay
    1. Warm up the spectrophotometer before use.
    2. Dilute unknowns if necessary to obtain between 5 and 100 g protein in at least
    one assay tube containing 100 l sample
    3. If desirred, add an equal volume of 1 M NaOH to each sample and vortex (see
    Comments below). Add NaOH to standards as well if this option is used.
    4. Prepare standards containing a range of 5 to 100 micrograms protein (albumin
    or gamma globulin are recommended) in 100 l volume. See how to set up an
    assay for suggestions as to setting up the standards.
    5. Add 5 ml dye reagent and incubate 5 min.
    6. Measure the absorbance at 595 nm.

    Analysis
    Prepare a standard curve of absorbance versus micrograms protein and determine
    amounts from the curve. Determine concentrations of original samples from the
    amount protein, volume/sample, and dilution factor, if any.

    Comments
    The dye reagent reacts primarily with arginine residues and less so with histidine,
    lysine, tyrosine, tryptophan, and phenylalanine residues. Obviously, the assay is less

    accurate for basic or acidic proteins. The Bradford assay is rather sensitive to bovine
    serum albumin, more so than "average" proteins, by about a factor of two.
    Immunoglogin G (IgG - gamma globulin) is the preferred protein standard. The
    addition of 1 M NaOH was suggested by Stoscheck (1990) to allow the solubilization
    of membrane proteins and reduce the protein-to-protein variation in color yield.

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