cytochromeb5（Cytochrome b5）Content Determination Kit Storybook

Micro method 100T/96S

Before formal testing, it is necessary to take 2-3 Make predictions based on samples with significant expected differencesMeasurement significance:

cytochromeP450 Enzymes are a group of isoenzymes mainly present in the liver, playing an important role in the metabolism of exogenous substances, especially drugs andMetabolism of toxins. cytochrome P450 And cytochromeb5 isP450 The change in the ratio of two heme proteins in the enzyme system is related to P450 Gratitude activitySexually closely related.

Measurement principle:

Oxidative cytochrome b5 After the reduction of Lianxia *, in 424nm There is a maximum absorption peak at this point, which can be measured by 424nm and 490nm The difference in absorbance values can be calculatedoutcytochromeb5The content.

Cytochrome B5 Content Determination Kit P450 Series

composition

Reagent 1: Powder×2 Bottle,4℃preservation. Add before use 100ml Distilled water, fully dissolved.

Preparation of working fluid:

Prepare before use, wear disposable gloves, carefully open the bottle cap of reagent three, and add reagent two 20ml Fully dissolved,4℃Can be stored in the dark 1 week

The specific product parameters are subject to the parameters in the product manual received

Self provided instruments and supplies:

Ordinary centrifuge, high-speed centrifuge, adjustable pipette, UV spectrophotometer/ELISA reader, micro glass colorimetric dish/96 Orifice plate and steamingDistilled water.

Cytochrome in the sample b5 extract：

1、Excluding large molecular substances such as nuclei and mitochondria:About it 0.5g Organize, join 1ml Reagent 1: Grind thoroughly on ice,10 000g 4℃leaveheart 30minTake the supernatant and transfer it into a high-speed centrifuge tube.

2、Crude microsomes:100 000g，4℃centrifugation 60minDiscard the supernatant.

3、Excluding impurities such as hemoglobin:Step by step 2 Add to the sediment 1ml Reagent 1, tightly cover and shake thoroughly to dissolve,100 000g centrifugation 30minAbandoningThe supernatant.

4、Liquid to be tested:Step by step 3 Add reagent 2 to the precipitate 0.5mlAfter sealing tightly, shake and dissolve thoroughly, which is the test liquid. The test liquid needs to be measured on the same day.

cytochrome b5 Content determination operation:

1. spectrophotometer/Preheating of ELISA reader 30 min.

2. Working fluid is placed in 25℃Preheating in a water bath 30 min.

3. Blank tube:取 1ml Glass colorimetric dish, add 10 μl distilled water200μl Working fluid, let stand at room temperature 2 min，424nm and 490nm Suck on the spotLight value,424nm The absorbance value is recorded asA Blank tube 1，490nm The absorbance value is recorded asA Blank tube 2.△A Blank tube=A Blank tube 1-A emptyWhite tube 2

4. Measurement tube:取 1ml Glass colorimetric dish, add 10 μl The liquid to be tested,200μl Working fluid, let stand at room temperature 2 min，424nm and 490nm Suck on the spotLight value,424nm The absorbance value is recorded asA Measurement tube 1，490nm The absorbance value is recorded asA Measurement tube 2.

△A Measurement tube=A Measurement tube 1-A Measurement tube 2.

AttentionJust need to make a blank tube.

Sample cytochrome b5 Content calculation formula:

a.The calculation formula for measuring using a trace quartz colorimetric dish is as follows

（1）Calculated by protein concentration

cytochromeb5 content(nmol/mg prot) = (△A Measurement tube-△A Blank tube)÷(ε×d)×V Anti General÷（Cpr×V sample）

=122.8×(△A Measurement tube-△A Blank tube)÷Cpr

（2）Calculated based on sample quality

cytochromeb5 Content（nmol/g Fresh weight)= (△A Measurement tube-△A Blank tube)÷(ε×d)×V Anti General×（V Sample General÷V sample）÷W

=61.4×(△A Measurement tube-△A Blank tube)÷W

εReduced Cytochromeb5 Namol extinction coefficient,171×10^-6 L/nmol/cm；dColor dish optical diameter（cm），1cm； V Counter total: the total volume of the reaction system,210 μl =2.1×10^-4 L；CprProtein concentration of the test solution（mg/ml）, requires additional testing;VSample: Add the volume of the test liquid to the reaction system,10 μl=0.01 ml；VSample total: the total volume of the liquid to be tested,0.5 ml；W: Sample quality（g）.

b.use 96 The calculation formula for orifice plate measurement is as follows

（1）Calculated by protein concentration

cytochromeb5 content(nmol/mg prot) = (△A Measurement tube-△A Blank tube)÷(ε×d)×V Anti General÷（Cpr×V sample）

=245.6×(△A Measurement tube-△A Blank tube)÷Cpr

（2）Calculated based on sample quality

cytochromeb5 Content（nmol/g Fresh weight)= (△A Measurement tube-△A Blank tube)÷(ε×d)×V Anti General×（V Sample General÷V sample）÷W

=122.8×(△A Measurement tube-△A Blank tube)÷W

εReduced Cytochromeb5 Namol extinction coefficient,171×10^-6 L/nmol/cm；d：96 Orifice plate optical diameter（cm），0.5cm； V Counter total: the total volume of the reaction system,210 μl =2.1×10^-4 L；CprProtein concentration of the test solution（mg/ml）, requires additional testing;VSample: Add the volume of the test liquid to the reaction system,10 μl=0.01 ml；VSample total: the total volume of the liquid to be tested,0.5 ml；W: Sample quality（g）.

Cytochrome B5 Content Determination Kit P450 Series