Application

The kit can detect the activity of Cu2+-ATPase in tissue, cultured cells and other samples.

Detection principle

ATPase exists on the cell membrane and organelle membrane and plays an important role in the transport of substances, energy conversion and information transmission. The body in the absence of O2 and some diseases conditions, the activity of ATPase take a series of changes. In addition some of the genetic disease have relation to the activity of the ATPase.

Detection principle

ATPase can decompose ATP to produce ADP and inorganic phosphorus. ATPase activity can calculated by measuring the content of inorganic phosphorus. Cu2+-ATPase is a kind of enzyme that can be activated by potassium and inhibited by vanadic acid.

Experiments instruments

Test tube, micropipettor, Vortex mixer, 37℃ water bath/gas bath, 45℃ water bath/gas bath, Low-speed centrifuge, Spectrophotometer (660 nm)

Sample pretreatment

Weigh the tissue accurately, add 9 times of normal saline at the ratio of Weight (g): Volume (mL) = 1:9. Make the mechanical homogenization in ice water bath to prepare 10% homogenate. Centrifuge at 3000 rpm for 10 min and take the supernatant for detection. Then dilute the 10% homogenate with normal saline at 1:4 to prepare 2% homogenate for detection. Meanwhile, determine the concentration of supernatant (E-BC-K318, E-BC-K168, E-BC-K165).

Operation steps

1.    Enzymatic reactions

Control tube (A)

Sample tube (B)

Reagent 1 (μL)

130

130

Reagent 2 (μL)

80

Reagent 3 (μL)

120

Reagent 4 working solution (μL)

40

40

Reagent 5 working solution (μL)

40

40

Reagent 6 working solution (μL)

40

Sample (μL)

100

Mix fully, incubate at 37℃ water bath for 10 min accurately. 

Reagent 7 (μL))

50

50

Sample (μL)

100

2.    Determination of Phosphorus

Simplified operation (for batch assay):

1.    Preparation of mixture solution A and B:

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

Control tube (A)

Sample tube (B)

Reagent 1 (μL)

130 × (n+2*)

130 × (n+2*)

Reagent 2 (μL)

80 × (n+2*)

Reagent 3 (μL)

120 × (n+2*)

Reagent 4 working solution (μL)

40 × (n+2*)

Reagent 5 working solution (μL)

40 × (n+2*)

40 × (n+2*)

Reagent 6 working solution (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 solution A and solution B, respectively.

2.    Simplified operation table:

(1) Enzymatic reaction:

Blank tube (A)

Sample tube (B)

A solution (μL)

330

B solution (μL)

330

Sample (μL)

100

Mix fully, incubate at 37℃ water bath for 10 min accurately.

Reagent 7 (μL))

50

50

Sample (μL)

100

Mix fully and centrifuge at 3500 rpm for 10 min, take 400 μL supernatant for phosphorus determination.

(2) Determination of Phosphorus

Notes

1.    This method has the advantages of trace-detection, sensitive and rapid. The tubes used in the detection are strictly required to avoid of any phosphorus contamination. It is recommended to use disposable plastic tubes to get rid of phosphorus contamination.

2.    The Phosphorus determination reagent cannot be stored for a long time. It is recommended to prepare fresh solution before use and the prepared solution must be use out in 24 h. Store the prepared solution in refrigerator at any time.

3.    It is recommended to prepare the mixture solution A and solution B in advance and operate the detection as the simplified operation, which is more quick and accurate.

4.    Containers for all of the reagents must be specified to avoid the contamination of phosphorus, including pipes for H2SO4 and container for water.