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Protein Determination Assays

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There is no completely satisfactory single method to ... Take the absorbance at 280 nm in a quartz cuvette ... Take absorption at 260 nm in a quartz cuvette ... – PowerPoint PPT presentation

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Title: Protein Determination Assays


1
Protein Determination Assays
  • Xin Li
  • Scott Group
  • 05/10/2005

2
Quantitative Determination of Proteins
  • There is no completely satisfactory single method
    to determine the concentration of protein in any
    given sample
  • The choice of the method depends on the nature of
    the protein, the nature of the other components
    in the protein sample, desired speed ,accuracy
    and sensitivity of assay

3
Methods Used for Protein Determination
  • Biuret Test
  • Folin-Ciocalteu ( Lowry ) Assay
  • Bicinchoninic Acid ( BCA ) Assay
  • Dye-Binding ( Bradford ) Assay
  • Ultraviolet Absorbance

4
Biuret Test
Peptide Chains
Biuret Complexes ( purple color )
  • Gornall, AG, CS Bardawill, and MM David. J. Biol.
    Chem. 177 751. 1949.
  • Layne, E. Spectrophotometric and Turbidimetric
    Methods for Measuring Proteins. Methods in
    Enzymology 10 447-455. 1957.
  • Robinson, HW and CG Hogden. J. Biol. Chem. 135
    707. 1940.
  • Slater, RJ (ed.). Experiments in Molecular
    Biology. Clifton, New Jersey Humana Press, 1986.
    P. 269.
  • Weichselbaum, TE. Am. J. Clin. Pathol. Suppl. 10
    40. 1946.

5
Biuret Test
  • Reproduciple
  • Very few interfering agents
  • (ammonium salts being one such agent )
  • Fewer deviations than with the Lowry or
    ultraviolet absorption methods
  • Requires large amounts protein (1-20mg)
  • Low sensitivity

6
Biuret Test
  • Warm up the spectrophotometer 15 min. before use.
  • Dilute samples to an estimated 1 to 10 mg/ml with
    buffer. Add 1 ml to each assay tube. Duplicate
    samples are recommended, and a range of dilutions
    should be used if the actual concentration cannot
    be estimated.
  • Prepare a reference tube with 1 ml buffer.
  • Prepare standards from 10 mg/ml bovine serum
    albumin, preferably calibrated using absorbance
    at 280 nm and the extinction coefficient. Range
    should be from 1 to 10 mg protein.
  • Add 9 ml Biuret reagent to each tube, vortex
    immediately, and let stand 20 min.
  • Read at 550 nm.

7
Folin-Ciocalteu ( Lowry ) Assay
  • Lowry, OH, NJ Rosbrough, AL Farr, and RJ Randall.
    J. Biol. Chem. 193 265. 1951.
  • Oostra, GM, NS Mathewson, and GN Catravas. Anal.
    Biochem. 89 31. 1978.
  • Stoscheck, CM. Quantitation of Protein. Methods
    in Enzymology 182 50-69 (1990).
  • Hartree, EF. Anal Biochem 48 422-427 (1972).

8
Folin-Ciocalteu ( Lowry ) Assay
  • Sensitive over a wide range
  • Can be performed at room temperature
  • 10-20 times more sensitive than UV detection
  • Can be performed in a microplate format
  • Many substances interfere with the assay
  • (Strong acids, ammonium sulfate )
  • Takes a considerable amount of time to perform
  • The assay is photosensitive, so illumination
    during the assay must be kept consistent for all
    samples
  • Amount of color varies with different proteins

9
Folin-Ciocalteu ( Lowry ) Assay
  • Add samples containing up to 100 µg of protein.
  • Bring all tubes to 1 mL total volume with water.
  • Prepare the Assay Mix and diluted Folin-Ciocalteu
    reagent.
  • To each tube add 5 mL of assay mix and thoroughly
    vortex.
  • Incubate tubes at room temperature for 10 min.
  • Add 0.5 mL of diluted Folin-Ciocalteu reagent.
    Vortex immediately.
  • Incubate at room temperature for 30 min.
  • Vortex the tubes, zero the spectrophotometer with
    the blank and measure absorbance at 500-750 nm.

10
Bicinchoninic Acid ( BCA ) Assay
  • P.K. Smith et al. (1985) Anal. Biochem. 150 76.
  • K. J. Wiechelman et al. (1988) Anal. Biochem.
    175 231

11
Bicinchoninic Acid ( BCA ) Assay
  • Very sensitive and rapid if you use elevated
    temperatures
  • Compatible with many detergents
  • Working reagent is stable
  • Very little variation in response between
    different proteins
  • Broad linear working range
  • The reaction does not go to completion when
    performed at room temperature

12
Bicinchoninic Acid ( BCA ) Assay
  • Prepare the required amount of protein
    determination reagent by adding 1 volume copper
    sulfate solution to 50 volumes of bicinchoninic
    acid solution.
  • Set up test tubes containing samples and known
    amounts of bovine serum albumin in the range of 0
    to 100 micrograms. Each tube should contain 0.1
    mL total volume.
  • Add 2.0 mL of the protein determination reagent
    to each tube and vortex.
  • Incubate the tubes at 60oC for 15 min.
  • Cool the tubes to room temperature and determine
    the absorbance at 562 nm.

13
Dye-Binding ( Bradford ) Assay
  • CBBG primarily responds to arginine residues
  • (eight times as much as the other listed
    residues)
  • If you have an arginine rich protein,
  • You may need to find a standard
  • that is arginine rich as well.
  • CBBG binds to these residues in the anionic form
  • Absorbance maximum at 595 nm (blue)
  • The free dye in solution is in the cationic form,
  • Absorbance maximum at 470 nm (red).
  • Bradford, MM. A rapid and sensitive for the
    quantitation of microgram
  • quantitites of protein utilizing the
    principle of protein-dye binding. Analytical
    Biochemistry 72 248-254. 1976.
  • Stoscheck, CM. Quantitation of Protein. Methods
    in Enzymology 182 50-69 (1990).

14
Dye-Binding ( Bradford ) Assay
  • Fast and inexpensive
  • Highly specific for protein
  • Very sensitive 1-20 µg (micro assay) 20-200 µg
    (macro assay)
  • Compatible with a wide range of substances
  • Extinction co-efficient for the dye-protein
    complex is stable over 10 orders of magnitude
    (assessed in albumin)
  • Dye reagent is complex is stable for
    approximately one hour
  • Non-linear standard curve over wide ranges
  • Response to different proteins can vary widely,
    choice of standard is very important

15
Dye-Binding ( Bradford ) Assay
  • Absorption spectra of anionic and cationic forms
    of the dye overlap.
  • So the standard curve is non-linear although all
    kit providers of the Bradford assay insist that
    the assay performs linearly.
  • The assay performs linearly over short
    concentration stretches.
  • If your sample is more than 20 micrograms, a
    second order curve will fit much better than a
    linear curve.

16
Dye-Binding ( Bradford ) Assay
  • Warm up the spectrophotometer for 15 min. before
    use
  • Dilute samples with buffer to an estimated
    concentration of 1 to 20 micrograms/milliliter
  • Prepare standards containing a range of 1 to 20
    micrograms protein (albumin or gamma globulin are
    recommended) to a volume of 200 µl (to a volume
    of 100 µl if you are adding 1 M NaOH)
  • Prepare unknowns to estimated amounts of 1 to 20
    micrograms protein per tube to 200 µl (100 µl if
    you are using 1 M NaOH)
  • Add 100 µl 1 M NaOH to each sample and vortex.
  • Add 800 µl dye reagent and incubate 5 min.
  • Measure the absorbance at 595 nm.

17
Ultraviolet Absorbance
  • If you don't know what the protein concentration
    of an unknown sample is likely to be, the
    ultraviolet method might be a good starting
    point.
  • This is often used to estimate protein
    concentration prior to a more sensitive method
  • Monitors the absorbance of aromatic amino acids,
    tyrosine and tryptophan
  • Higher orders of protein structure, many other
    cellular components, and particularly nucleic
    acids, also may absorb UV light
  • This method is the least sensitive of the methods
  • The real advantages of this method are that the
    sample is not destroyed and that it is very
    rapid.
  • Layne, E. Spectrophotometric and Turbidimetric
    Methods for Measuring Proteins. Methods in
    Enzymology 3 447-455. 1957.
  • Stoscheck, CM. Quantitation of Protein. Methods
    in Enzymology 182 50-69. 1990.

18
Ultraviolet Absorbance
  • Quick
  • Sample can be recovered
  • Useful for estimation of protein before using a
    more accurate method
  • Highly susceptible to contamination by buffers,
    biological materials and salts
  • Protein amino acid composition is extremely
    important, thus the choice of a standard is very
    difficult, especially for purified proteins
  • Absorbance is heavily influence by pH and ionic
    strength of the solution.

19
Ultraviolet Absorbance
Estimation Procedure
  • Zero spectrophotometer to water (or buffer)
  • Take the absorbance at 280 nm in a quartz cuvette
  • Change wavelength to 260 nm and zero with water
    (or buffer)
  • Take absorption at 260 nm in a quartz cuvette
  • Use the following equation to estimate the
    protein concentration
  • Protein (mg/mL) 1.55A280 0.76A260

20
BioRad DC Protein Assay
  • Based on Lowry Assay with following improvements
  • Reaches 90 of its maximum color development
    within 15 minutes
  • The color changes not more than 10 in 2 hours

21
BioRad DC Protein Assay
  • Prepare 5 dilutions of samples and 5 dilutions of
    a protein standard containing from 0.2 mg/ml to
    about 1.5 mg/ml protein. A standard curve should
    be prepared each time the assay is performed. For
    best results, the standard should be prepared in
    the same buffer as the sample.

22
BioRad DC Protein Assay
  • Pipet 5 ul of standards and samples into a
    microtiter plate
  • Add 25ul of reagent A into each well
  • Add 200ul reagent B into each well
  • Agitate the plate to mix the reagents

Empty Air
BlankReagent AB
0Reagent AB5ul water
23
BioRad DC Protein Assay
  • After 15 minutes, absorbance can be read at
    750nm. The absorbance will be stable for about 1
    hour

From Oliver
24
BioRad DC Protein Assay
From Microplate reader in Bioexpress Lab
25
BioRad DC Protein Assay
Plot Concentration as Y axis
Plot Concentration as X axis
VS
y 19.815x - 0.2861
y 0.0498x 0.015
Protein 19.815A- 0.2861
Protein(A -0.0015)/0.0498
Could introduce errors into the calculation
26
BioRad DC Protein Assay
Protein 19.815A- 0.2861
Protein Concentration 3.1661mg/ml
27
Tips
  • Use clean glassware and supplies
  • Make sure cuvettes are clean of all residues
  • Protein assays are strongly influenced by the
    composition of the proteins present in your
    sample
  • Become familiar with spectrophotometry before
    proceeding
  • Always let a spectrophotometer warm up for 15-20
    minutes before using
  • Know the limits of the spectrophotometer with
    which you are using

28
Tips
  • Standard curves are not always linear
  • The protein used for your standard curve must
    make sense
  • Make sure your standard curve covers the
    absorbance range of your unknown with at least
    two points on either side
  • Make sure that your protein solution behaves in a
    reproducible manner to the assay method by making
    a dilution curve
  • Use buffer and water blanks to anchor down your
    standard curve
  • Place the protein concentration on the y-axis of
    you standard curve plot so that you can use the
    best-fit equation directly for concentration
    determination
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