​Application

This kit can be used for detection of total-ATPase activity in animal erythrocyte, whole blood and other samples. This kit (100 Assays) can detect 48 samples.

Detection significance

ATPase exists on the membrane of tissue cells and organelles. It is a kind of protease on the biological membrane which plays an important role in material transport, energy conversion and information transmission. The enzyme activity of ATPase will have a series of changes when the body in the hypoxic or diseases condition, and is also associated with some genetic diseases.

Detection principle

ATPase can decompose ATP into ADP and inorganic phosphorus. ATPase activity can be calculated by determining the content of inorganic phosphorus. 

Experimental instruments

Test tube, Micropipettor, Vortex mixer, 37℃ water bath, Spectrophotometer (636 nm)

Sample pretreatment

1.   Erythrocyte:

Method 1: take heparin anticoagulant whole blood and centrifuge for 10 min at 1000 rpm. Discard the supernatant and keep the erythrocyte sedimentation, then add 49 times the volume of double distilled water, mix fully to get hemolysis (the liguid is transparent to light).

Method 2: take heparin anticoagulant whole blood and add double distilled water at a volume ratio of whole blood: add double distilled water = 1:24, mix fully to prepare 25 times diluted hemolysis (the liguid is transparent to light).

2.   Tissue:

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 2500 rpm for 10 min and take the supernatant (10% homogenate). Then dilute the 10% homogenate with normal saline for 10 times to prepare 1% homogenate for detection. Meanwhile, determine the protein concentration of supernatant (E-BC-K318-M, E-BC-K168-S, E-BC-K165-S). If the result of pre-experiment is too high, dilute the 1% tissue homogenate to different concentrations for pre-experiment and determine the sample concentration according to the result.

3.   Culture cell samples:

Collect and centrifuge the culture cells, discard the supernatant and keep the cell sediment. Add 0.2~0.3 mL of normal saline or homogenate medium to prepare 107/mL cell suspension, then broken the cells by homogenizer, ultrasonic crusher or freezing/thawing cycles. The prepared cell suspension does not need to be centrifuged. Meanwhile, determine the protein concentration of supernatant (E-BC-K318-M, E-BC-K168-S, E-BC-K165-S).Then dilute the cell homogenate to different concentrations for pre-experiment and determine the sample concentration according to the result.

[Note] 1. The sample must be mix fully when test the sample.

2. Do not use phosphate buffer or phosphorus-containing reagents as homogenization media or dilute samples.

3. The absolute OD value (the OD value of sample- the OD value of control) in pre-experiment should be control about 0.2.

4. The prepared hemolysis should be detect as soon as possible, otherwise the enzyme in the sample is susceptible to inactivation.

5. The enzyme activity will be affect when treat the cells by freezing/thawing cycles.

Operation steps

1.  Enzymatic reactions

2.  Determine phosphorus

Note: the cuvette should be rinse with running water for 10 times and then wash with double distilled water for 4~5 times. It will avoid the contamination of phosphorus.

Notes

1.  This kit is for research use only.

2.  Please progress strictly with operation procedures.

3.  Do not use components from different batches of kit.

4.  The valid period of kit is 6 months and the expiration date is on the packing box.

5.  If the ATPase activity is calculated by erythrocyte, the number of erythrocyte in sample needs to be counted separately.

6.  If the ATPase activity is calculated by hemoglobin concentration, hemoglobin concentration of the sample needs to be determined separately.

7.  It is recommended to use disposable tubes or new tubes to avoid pollution of phosphorus which is the key for success.