| Usage | 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.
1. 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.
2. 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.
3, 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:
1. The microplate reader or visible light spectrophotometer was preheated for more than 30 min, and the wavelength was adjusted to 510 nm.
2. 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 | | 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. |
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. |
| General Notes | 1. This product is only for scientific research use, not for clinical diagnosis. See the Safety Data Sheet for hazard and safe handling information.
2, do not mix components between different batch numbers and different manufacturers; Otherwise, it may lead to abnormal results.
3, when mixing or redissolving the components, avoid air bubbles.
4, frequently change the suction head to avoid cross contamination between the components.
5, before the start of the experiment, ensure that all the components and equipment in the appropriate temperature.
6, Chromogen and Chromogen B and Chromogen C all needs to avoid light preservation. Chromogen C cannot be used again after it turns bluish-green.
7. After adding Chromogen C, it must be mixed immediately, otherwise the color will not be complete. |
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