Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Laboratory
Studies
Overview
Microscope studies
Flagella experiment
Laboratory math
Blood fractionation
Gel electrophoresis
Protein gel analysis
Mitochondria
Concepts/ theory
Recordkeeping, Writing,
& Data Analysis
Laboratory
Methods
Overview
Keeping a lab notebook
Writing research papers
Dimensions & units
Using figures (graphs)
Examples of graphs
Experimental error
Representing error
Applying statistics
Overview
Principles of microscopy
Solutions & dilutions
Protein assays
Spectrophotometry
Fractionation & centrifugation
Radioisotopes and detection
List of methods
Direct absorbance
measurement
absorbance at 280 nm
absorbance at 205 nm
extinction coefficient
Colorimetric assays
set up an assay
spectrophotometry
modified Lowry
biuret
Bradford
Bicinchoninic Acid
(Smith)
Principle
Under alkaline conditions the divalent copper ion forms a complex with peptide bonds in which it is reduced to a monovalent ion. Monovalent copper ion and the radical
groups of tyrosine, tryptophan, and cysteine react with Folin reagent to produce an unstable product that becomes reduced to molybdenum/tungsten blue.
Equipment
In addition to standard liquid handling supplies a spectrophotometer with infrared lamp and filter is required. Glass or polystyrene (cheap) cuvettes may be used.
Assay
1. Prepare a series of dilutions of 0.3 mg/ml bovine serum albumin in the same buffer containing the unknowns, to give concentrations of 30 to 150 micrograms/ml
(0.03 to 0.15 mg/ml).
2. Add 1.0 ml each dilution of standard, protein-containing unknown, or buffer (for the reference) to 0.90 ml reagent A in separate test tubes and mix.
3. Incubate the tubes 10 min in a 50 degrees C bath, then cool to room temperature.
4. Add 0.1 ml reagent B to each tube, mix, incubate 10 min at room temperature.
5. Rapidly add 3 ml reagent C to each tube, mix, incubate 10 min in the 50 degree bath, and cool to room temperature. Final assay volume is 5 ml.
6. Measure absorbance at 650 nm in 1 cm cuvettes.
Analysis
Prepare a standard curve of absorbance versus micrograms protein (or vice versa), and determine amounts from the curve. Determine concentrations of original samples
from the amount protein, volume/sample, and dilution factor, if any.
Assay
1. Dilute samples to an estimated 0.025-0.25 mg/ml with buffer. If the concentration can't be estimated it is advisable to prepare a range of 2-3 dilutions spanning an
order of magnitude. Prepare 400 microliters each dilution. Duplicate or triplicate samples are recommended.
2. Prepare a reference of 400 microliters buffer. Prepare standards from 0.25 mg/ml bovine serum albumin by adding 40-400 microliters to 13 x 100 mm tubes + buffer
to bring volume to 400 microliters/tube.
3. Add 400 microliters of 2x Lowry concentrate, mix thoroughly, incubate at room temp. 10 min.
4. Add 200 microliters 0.2 N Folin reagent very quickly, and vortex immediately. Complete mixing of the reagent must be accomplished quickly to avoid
decomposition of the reagent before it reacts with protein. Incubate for 30 min. more at room temperature.
5. Use glass or polystyrene cuvettes to read the absorbances at 750 nm. If the absorbances are too high, they may be read at 500 nn.
Comments
Recording of absorbances need only be done within 10 min. of each other for this modified procedure, whereas the original Lowry required precise timing of readings due
to color instability. This modification is less sensitive to interfering agents and is more sensitive to protein than the original. As with most assays, the Lowry can be scaled
up for larger cuvette sizes, however more protein is consumed. Proteins with an abnormally high or low percentage of tyrosine, tryptophan, or cysteine residues will give
high or low errors, respectively.
References
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).