The Lowry Assay (for protein concentration)
How does it work?
- The first step is a Biuret reaction which reduces Cu+2 to Cu+1
- The
second reaction uses Cu+1 to reduce the Folin-Ciocalteu reagent (phosphomolybdate and phosphotungstate). This is detectable
in the range of 500 to 750 nm
Detection Limitations
Advantages
- Sensitive over a wide range
- The most commonly referenced procedure
for protein determination
- Can be performed at room temperature
- 10-20 times more sensitive than UV detection
- Can be performed in a
microplate format
Disadvantages
- Many substances interfere with the assay
- Alkaline copper reagent is laborious to prepare and will develop
carbonate scales over storage which interfere with optical activity, thus it must be prepared fresh daily
- 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
General Considerations
- Some researchers have reported that repeated assays in the same cuvettes
cause them to be etched
- Many chemical distributors sell a modified Lowry assay that is more stable and sensitive than homemade versions
- Since reduced copper is detected in the procedure, make sure that the distilled water used in the procedure is fed from plastic lines
and not copper lines. In general water from 18 megaohm water polishers is satisfactory
- Variation in the content of tyrosine
and tryptophan residues will influence the assay
Procedure
Alkaline Reagent
0.1 M NaOH
2% Na2CO3
0.5% Na Tartrate (use of
potassium salt will cause SDS to be insoluble)
0.5% Na Dodecylsulfate
Copper Reagent
1% CuSO4*5H2O
Assay Mix (MAKE FRESH EACH DAY)
50 mL
alkaline reagent and 0.5 mL copper reagent
Folin-Ciocalteu Reagent
Dilute with an equal volume of water to prepare the desired volume
Procedure:
- Add
samples containing up to 100 μg of protein
- Bring all tubes to 1 mL total volume with water. Be sure to have two tubes containing
only water as blanks. Also use reagent or buffer blanks if needed.
- 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 660 nm (or other appropriate wavelength). The data from the standard curve are usually linear
enough that a straight-line interpolation can be used to determine the concentration of unknowns.