BioProtocols--western5

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

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

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

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 ¦جg 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.