Glucose-6-Phosphate Dehydrogenase (G6PD) Activity Assay Kit #12581

#12581 Glucose-6-Phosphate Dehydrogenase (G6PD) Activity Assay Kit Protocol

A. Solutions and Reagents

NOTE: Prepare solutions with deionized/purified water or equivalent.

1. Tris Assay Buffer: Bring to room temperature before use.
2. G6PDH Substrate (40X): Reconstitute lyophilized component with 250µl dH₂O.
3. G6PDH Developer (100X): Reconstitute lyophilized component with 100µl dH₂O.
4. G6PDH Positive Control (100X): Reconstitute lyophilized component with 50µl dH₂O.
5. NADP+ (100X): Reconstitute lyophilized component with 100µl dH₂O.
6. G6PDH Cofactor (100X): Reconstitute lyophilized component with 100µl dH₂O.
7. PathScan® Sandwich ELISA Lysis Buffer (1X) (#7018): Thaw and keep on ice.

Working Solution Preparation

1. Calculate the number of tests: number of samples + positive controls.
2. Dilute Total Detection Solution for the calculated number of tests.
   1. 70 µl/well is recommended for a 96-well plate.
   2. 20 µl/well is recommended for a 384-well plate.

   Example: Number of tests = 10 samples (n=3) + 3 positive controls

   1. 96-Well Plate: 70 µl/well x 33 samples = 2310 µl + 10% = ~2500 µl
   2. 384-Well Plate: 20 µl/well x 33 samples = 660 µl + 10% = ~750 µl
3. Make Negative Control Solution (See Table).

   Note: DO NOT add G6PD substrate to the Negative Control Solution.

4. Make Positive Control Solution (See Table).

   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)	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
   Tris Assay Buffer	2362.5	320	99
   Total (µl) (with ~10% extra volume)	2500 (30 samples + 3 positive controls)	330	100

   Table 2: Example calculation for 10 samples in a 384-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	0
   Tris Assay Buffer	708.5	97	99
   Total (µl) (with ~10% extra volume)	750 (30 samples + 3 positive controls)	100	100

B. 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.

C. Test Procedure

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 15-30 min.
6. Read RFU on a plate reader with excitation around 540 nm and emission around 590 nm.