BMBMethod:lowry

From OpenWetWare

Jump to: navigation, search
OldMain2.jpg


Modified Lowry (room temperature w/SDS)

  1. Prepare 400 μL of sample dilution (estimated 0.025-0.5 mg/ml). Duplicate or triplicate samples are recommended.
  2. Add 400μL of 2x Lowry concentrate, mix thoroughly
  3. Incubate at room temp. 10 min.
  4. Add 200μL 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.
    1. Commercial Folin reagent is usually at 2N, so you will likely need to dilute 10-fold
  5. Incubate for 30 min., or more, at room temperature.
  6. Use glass or polystyrene cuvettes to read the absorbances at 750 nm. If the absorbances are too high, they may be read at 500 nm.


Notes

  • 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. The color is lost at a rate of approximately 1% per hour for the first few hours.
  • 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.
  • Similarly, the assays can be scaled down for use in a 96-well plate. Tien lab regularly does lowrys in this way at 1/5 the volume listed in the protocol (200μL assays)
  • We generally set up standard curves from 5-150 ug BSA per 1mL assay rxn


Reagents

Stocks

  • 5% Sodium Carbonate
  • 1% (w/v) cupric sulfate (5x hydrated)
  • 2% sodium potassium tartrate (w/v)
  • 10% sodium dodecyl sulfate (SDS)
  • 1 M NaOH


2x Lowry (made from above stock solutions)

ReagentAmt FinalAmt in Assay
20 parts Sodium Carbonate2.5%1%
1 part Copper0.025%0.01%
1 part tartrate0.05%0.02%
8 parts NaOH200mM80mM
10 parts SDS2.5%1%











Warm the solution to 37°C if a white precipitate forms, and discard if there is a black precipitate.

References

Peterson, G. L. (1977). A simplification of the protein assay method of Lowry et al. which is more generally applicable. Analytical Biochemistry, 83(2), 346–356. doi:10.1016/0003-2697(77)90043-4

Personal tools