Self-supplied consumables:
Microplate reader or visible light spectrophotometer (can measure the absorbance at 510 nm)
Thermostatic box, ice machine, low temperature centrifuge
96-well plate or microglass cuvette
Adjustable pipet gun and tip
deionized water
Homogenizer (if it is a tissue sample)
Reagent preparation
Extraction Buffer
read-to-use type; Before use, equilibrate to room temperature; Store at 4 ° C.
Chromogen A
ready-to-use; Before use, equilibrate to room temperature; Store away from light at 4 ° C.
Chromogen B
ready-to-use type; Before use, equilibrate to room temperature; Store away from light at 4 ° C.
Chromogen C
ready-to-use type; Before use, equilibrate to room temperature; Store away from light at 4 ° C.
Standard
ready-to-use type; Before use, balance to room temperature; Store at 4 ° C.
Sample preparation
Note
Fresh samples are recommended, and if experiments are not performed immediately, samples can be stored at -80 ° C 1 month.
- Tissue samples: Weigh about 0.1 g sample, add 1 mL Extraction Buffer, homogenize in an ice bath, centrifuge at 10,000 rpm for 10 min at 4 ° C, take the supernatant and place it on ice for testing.
- Serum (plasma) : plasma and serum can be directly used for determination. EDTA and citrate can not be used in plasma preparation, but other anticoagulants can be used.
- urine: direct measurement.
Note
To measure protein concentration, use Item No. : abs9232 ? protein quantification kit (BCA method) was used to determine the protein concentration of samples.
Experimental procedures
- The microplate reader or visible light spectrophotometer was preheated for more than 30 min, and the wavelength was adjusted to 510 nm.
- the table below for sample and response:
| Reagent | Blank tube (μL) | Standard tube (μL) | Control ?tube (μL) | Detector tube (μL) |
|---|
| Deionized water | 4 | 0 | 0 | 0 |
| Standard | 0 | 4 | 0 | 0 |
| Sample | 0 | 0 | 0 | 4 |
| Chromogen A | 40 | 40 | 40 | 40 |
| Chromogen B | 40 | 40 | 40 | 40 |
| After blending at 37 ℃ for 15 min incubation | After blending at 37 ℃ for 15 min incubation | After blending at 37 ℃ for 15 min incubation | After blending at 37 ℃ for 15 min incubation | After blending at 37 ℃ for 15 min incubation |
| Chromogen C | 120 | 120 | 120 | 120 |
| Sample | 0 | 0 | 4 | 0 |
| After blending 510 nm absorbance, the standard and need to make A blank, all needs to control each sample, the hole of the absorbance were recorded as A blank, A standard, A comparison, A measurement. | After blending 510 nm absorbance, the standard and need to make A blank, all needs to control each sample, the hole of the absorbance were recorded as A blank, A standard, A comparison, A measurement. | After blending 510 nm absorbance, the standard and need to make A blank, all needs to control each sample, the hole of the absorbance were recorded as A blank, A standard, A comparison, A measurement. | After blending 510 nm absorbance, the standard and need to make A blank, all needs to control each sample, the hole of the absorbance were recorded as A blank, A standard, A comparison, A measurement. | After blending 510 nm absorbance, the standard and need to make A blank, all needs to control each sample, the hole of the absorbance were recorded as A blank, A standard, A comparison, A measurement. |
The results were calculated as follows:
Note
We provide you with the calculation formula, including the derivation process calculation formula and the concise calculation formula. The two are exactly the same. The concise formula in bold is recommended as the final formula.
1, according to the protein concentration calculation
Active unit definition
37 ℃ per minute per mg protein catalytic produce 1 mu mol of phenol as a unit of enzyme activity. AKP/ALP (U/mg prot) = [C standard x (A determination - A contrast) present sample (A standard - A blank) * V] present sample (Cpr) V) present T = 0.133 x (A determination - A contrast) present (A standard - A blank) present Cpr
2, fresh weight calculation according to the sample
Active unit definition
37 ℃ in the organization to make 1 mu per minute per gram mol of phenol as a unit of enzyme activity.
AKP/ALP (U/g fresh weight) = [C standard x (A determination - A contrast) present sample (A standard - A blank) * V] present (W present V sample extraction (V) present T = 0.133 x (A determination - A contrast) present (A standard - A blank) present W
3, according to the liquid volume calculation
One unit of activity was defined as the catalytic production of 1 μmol phenol per minute per milliliter of blood or urine at 37 ° C.
AKP/ALP (U/mL) = [C standard x (A determination - A contrast) present sample (A standard - A blank) * V] present V sample present T = 0.133 x (A determination - A contrast) present (A standard - A blank)
C standard
concentration of standard, 2 μmol/mL; V: join the upper clear liquid deposition reaction system, 0.004 mL; T: reaction time, 15 min; V extraction: Add extraction liquid volume, 1 mL; W: fresh weight of sample, g; Cpr: the protein concentration in the supernatant fluid, mg/mL.
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