H+K+-ATPase is a member of the P-type ATPase family and mediates the exchange and transport of intracellular hydrogen ions and extracellular potassium ions. Gastric H+K+-ATPase (HKAg) mainly exists in gastric mucosal wall cells and a small amount in renal medulla. As a membrane-bound protein, HKAg has the function of acidifying gastric contents and activating pepsin, and can be used as a therapeutic target for peptic ulcer disease. However, colonic HKA (HKAc) exists in the colon or other tissues and mediates the reabsorption of active K. 

1.   The tubes used in assay must be disposed strictly without a trace of phosphorus. It is better to use disposable tubes or new tubes to avoid pollution of phosphorus which is the key for success.

2.   All the containers of reagents should be dedicated, including the pipette of drawing sulfuric acid and distilled water containers.

3.   The protein concentration of the sample to be tested should be less than 3 mg/mL.

1.  Preparation of working solution A and B:

Prepare needed amount of the fresh working solution A (for control tube) and working solution B (for sample tube) according to the following table.

Working solution A 

Working solution B 

Reagent 1 (μL)

130 × (n+2*)

130 × (n+2*)

Reagent 2 (μL)

80 × (n+2*)

Reagent 3 (μL)

120 × (n+2*)

Reagent 4 application solution (μL)

40 × (n+2*)

40 × (n+2*)

Reagent 5 application solution (μL)

40 × (n+2*)

40 × (n+2*)

Reagent 6 (μL)

40 × (n+2*)

Total amount of mixture reagent (μL)

330 × (n+2*)

330 × (n+2*)

[Note] n refers to the number of sample.

2*: Prepare 2 more tubes of working solution A and working solution B, respectively.

2.  Operation procedure

1)  Enzymatic reaction

(1)  Control tube: take 330 μL of working solution A to 1.5 mL EP tube.

Sample tube: take 330 μL of working solution B to 1.5 mL EP tube.

(2)  Add 100 μL of sample to sample tube.

(3)  Mix fully and incubate at 37℃ for 10 min.

(4)  Add 50 μL of reagent 7 application solution to each tube.

(5)  Add 100 μL of sample to control tube.

(6)  Mix fully and centrifuge at 2000 g for 10 min, take 400 μL supernatant of each tube for phosphorus assay.

2)  Phosphorus assay

(1)  Standard tube: take 400 μL of 0.5 μmol/mL standard to 5 mL EP tube

Control tube: take 400 μL of supernatant from corresponding sample tube to 5 mL EP tube.

Sample tube: take 400 μL of supernatant from corresponding sample tube to 5 mL EP tube.

(2)  Add 2000 μL of phosphorus assay reagent to each tube.

(3)  Mix fully, incubate at 45℃ for 10 min and cool to room temperature.

1)      Enzymatic reaction

2)      Phosphorus assay