A. Instrumentation

  1. A plate reader that can read 96-well plates with excitation around 530 nm, and emission at 590 nm.
  2. 96-well black plate.

B. Reagent Preparation

  1. Please reconstitute the follow lyophilized components:
    Component dH2O Volume
    G6PDH Substrate 250 μL
    G6PDH Developer 100 μL
    G6PDH positive control 50 μL
    NADP 100 μL
    G6PDH cofactor 100 μL
  2. Calculate the Number of Tests: number of samples + positive controls.
  3. Dilute Total Detection Solution for the calculated Number of Tests.
    • 70 μL/well is recommended for a 96-well plate
    • 20 μL/well is recommended for a 384-well plate.
  4. Number of Tests = 10 samples (n=3) + 3 positive controls

    • 96-Well Plate: 70 μL/well x 33 samples = 2310 μL + 10% = ~2500 μL
    • 384-Well Plate: 20 μL/well x 33 samples = 660 μL) + 10% = ~750 μL
  5. Make Negative Control Solution (See Table)
  6. Note: DO NOT add G6PD substrate to the Negative Control Solution.

  7. Make Positive Control Solution (See Table)
  8. Make 1X Cell lysis buffer
  Total Detection Solution (μL) Negative Control Solution (μL) Positive Control Solution (μL)
G6PD Substrate (40X) 62.5 0 0
G6PD Developer (100X) 25 3.3 0
G6PDH cofactor (100X) 25 3.3 0
G6PD positive control (100X) 0 0 1
NADP+ (100X) 25 3.3 0
G6PD Assay Buffer (1X) 2362.5 320 99
Total (μL) (With ~10% extra volume) 2500 (30 Samples + 3 Positive Controls) 330 100

Table 1: Example of Calculation for 10 Samples in a 96-well plate (All triplicates)

  Total Detection Solution (μL) Negative Control Solution (μL) Positive Control Solution (μL)
G6PD Substrate (40X) 19 0 0
G6PD Developer (100X) 7.5 1 0
G6PDH cofactor (100X) 7.5 1 0
G6PD positive control (100X) 0 0 1
NADP+ (100X) 7.5 1  
G6PD Assay Buffer (1X) 708.5 97 99
Total (μL) (With ~10% extra volume) 750 (30 Samples + 3 Positive Controls) 100 100

Table 2: Example Calculation for 10 samples in a 384-plate (All triplicates)

C. Preparing Cell Lysates

For adherent cells

  1. Grow target cells to 80–90% confluence and aspirate media.
  2. Add fresh media containing regulator for desired time.
  3. Aspirate media and rinse cells once with ice-cold 1X PBS (#9872).
  4. Aspirate PBS and add 0.5 ml ice-cold 1X Cell Lysis Buffer plus 1 mM PMSF to each plate (10 cm diameter).
  5. Incubate the plate on ice for 5 min.
  6. Scrape cells off the plate and transfer to an appropriate tube. Keep on ice.
  7. Sonicate lysates on ice.
  8. Microcentrifuge for 10 min (x14,000 rpm) at 4°C and transfer the supernatant to a new tube. The supernatant is the cell lysate.
  9. Store at −80°C in single-use aliquots.

For suspension cells

  1. Remove media by low speed centrifugation (~1200 rpm) when the culture reaches 0.5–1.0 x 106 viable cells/ml. Treat cells by adding fresh media containing regulator for desired time.
  2. Collect cells by low speed centrifugation (~1200 rpm) and wash once with 5–10 ml ice-cold 1X PBS.
  3. Cells harvested from 50 ml of growth media can be lysed in 2.0 ml of 1X Cell Lysis Buffer plus 1 mM PMSF.
  4. Sonicate lysates on ice.
  5. Microcentrifuge for 10 min (x14,000 rpm) at 4°C and transfer the supernatant to a new tube. The supernatant is the cell lysate. Store at −80°C in single-use aliquots.

D. Assay Protocol

  1. Dilute sample in G6PD Assay Buffer (1X) to desired concentration.
  2. Add 70 μL of Total Detection Solution and 30 μL of sample in a black 96-well plate. Mix well. (Alternatively, add 20 μL of Total Detection Solution and 10 μL of sample for a 384-well plate.)
  3. Negative control: Add 100 μL of Negative Control Solution to three wells.(Add 30 μL of Negative Control Solution for a 384-well plate.)
  4. Positive control: Add 70 μL of Total Detection Solution and 30 μL Positive Control to three wells. Mix well. (Add 20 μL of Total Detection Solution and 10 μL of Positive Control for a 384-well plate.)
  5. Incubate at 37°C for 15 minutes.
  6. Read RFU on a plate reader with excitation around 540 nm and emission around 590 nm.

posted November 5, 2013