LEAP Testing Service

LOWRY WITH PRECIPITATION (L005)

I. Scope
This total protein assay measures the level of protein in latex extracts after a protein precipitation step to decrease assay interferences. This procedure paraphrases method ASTM D 5712-05.

II. Procedure
The assay is performed according to the microassay protocol in a 96-well plate. Samples are serially diluted 4X, and each dilution and all standards are done in duplicate. Each plate can hold one set of standards and 10 test samples.

III. Steps
1. Preparation of Oval Albumin Standard

• Mix 320 ul of 1 mg/ml oval albumin standard with 1680 ul of deionized water (160 ug/ml standard).
• Make seven twofold serial dilutions (1.0 ml to 1.0 ml water) in microfuge tubes.
• Concentration in ug/ml is as follows: 160, 80, 40, 20, 10, 5, 2.5 and water blank.

Comments: Because of the 4X concentration step in the assay, the standards will range from 640 to 10 ug/ml.

2. Protein Precipitation

• Place 1.0 ml of sample or standard in 1.5 ml microfuge tube.
• Add 100 ul of 0.15% (w/v) Sodium deoxycholate (DOC) vortex and let stand 10 minutes at room temperature.
• Add 100ul of 72% (w/v) trichloroacetic acid (TCA).
• Add 100 ul of 72% (w/v) phosphotungstic acid (PTA), vortex and let stand 20 minutes at room temperature.
• Centrifuge for 20 minutes at 6000 xg.
• Decant supernatant and resuspend pellet in 250 ul of 0.2 M NaOH.

Comments: Precipitates protein and allows for removal of interfering substances. If pellet does not resuspend in 250 ul NaOH place tube in sonicator bath. If pellet still fails to dissolve add another 250 ul NaOH and mix. This will change the starting dilution of this sample, adjust calculations accordingly.

3. Lowry Assay in Microplate

• Add 60 ul 0.1 M NaOH to wells B3-B12 through D3-D12 and F3-F12 through H3-H12. Add 60 ul of:
  
  • standard (Wells A1,A2 [160 ug/ml] through G1,G2 [10 ug/ml ]) H1 and H2 are NaOH blank, or
  • samples (Wells A3-A12, B3-B13 and E3-E12, F3-F12)
• Serially dilute test samples and discard 25 ul left after mixing rows D and H.
• Place 125 ul of alkaline copper tartrate (Reagent A) into each well.
• Add 15 ul of Folin reagent into each well (Reagent B).
• Allow to react for 10 minutes then read absorbance at 700 nm after 15 to 30 minutes.

Comments: DO NOT carry sample from row D to row E as this is the first well of another sample! NOTE: Glove Extracts—Test as is. For films or samples high in protein content a 1:4 dilution prior to precipitating should be sufficient. Liquid Latex—Dilute 10 ul with 990 ul of water, centrifuge for 5 minutes at 6000 xg, then transfer 500 ul to a clean tube for Lowry assay.