Protein Determination Assays

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Protein Determination Assays

• Xin Li
• Scott Group
• 05/10/2005

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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

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Methods Used for Protein Determination

• Biuret Test
• Folin-Ciocalteu ( Lowry ) Assay
• Bicinchoninic Acid ( BCA ) Assay
• Dye-Binding ( Bradford ) Assay
• Ultraviolet Absorbance

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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.

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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

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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.

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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).

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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

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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.

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Bicinchoninic Acid ( BCA ) Assay

• P.K. Smith et al. (1985) Anal. Biochem. 150 76.
• K. J. Wiechelman et al. (1988) Anal. Biochem.
  175 231

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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

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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.

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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).

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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

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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.

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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.

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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.

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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.

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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

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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

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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.

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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
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BioRad DC Protein Assay

• After 15 minutes, absorbance can be read at
  750nm. The absorbance will be stable for about 1
  hour

From Oliver
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BioRad DC Protein Assay
From Microplate reader in Bioexpress Lab
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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
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BioRad DC Protein Assay
Protein 19.815A- 0.2861
Protein Concentration 3.1661mg/ml
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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

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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