BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
羟脯氨酸（HYP）是一种非必需氨基酸，机体内胶原组织的主要成分之一，除弹性蛋白含少量羟 脯氨酸外，羟脯氨酸大多存在于胶原中，羟脯氨酸含量可作为衡量机体胶原组织代谢的重要指标，并对组织纤维化程度的判定具有重要意义。
检测原理：待测样本经酸水解产生游离羟脯氨酸，被氯胺T氧化为吡咯，吡咯能够与对二甲氨基苯甲醛 反应生成红色化合物，产物在560nm处具有特征吸收峰，通过吸光值的变化即可定量检测羟脯氨酸的含量。
适用样本：动物组织、细菌和培养细胞、血清或血浆等
注意：正式检测前，建议选择2-3个预期差异较大的样本进行预实验。
自备仪器和试剂
操作步骤
1. 试剂准备
| 试剂名称 | 注意事项 | 储存 |
| Tissue Extraction Buffer （自备） | 即用型；6 mol/L 盐酸，浓盐酸：去离子水（V/V）=1:1 | 室温保存 |
| Cell Extraction Buffer | 即用型； 使用前平衡到室温 | 4℃保存 |
| 试剂一 | 即用型； 使用前平衡到室温 | 4℃避光保存 |
| 试剂二 | 即用型； 使用前平衡到室温 | 4℃避光保存 |
| 标准品 | 即用型； 0.5mg/mL L-羟基脯氨酸标准品 使用前平衡到室温 | 4℃避光保存 |
2. 标准品制备
使用0.5 mg/mL L-羟基脯氨酸标准品，按照下表所示，进一步稀释成标准品：
| 序号 | 标准品体积（μL） | 去离子水体积（μL） | 浓度（μg/mL） |
| Std.1 | 24 μL 0.5 mg/mL Standard | 376 | 30 |
| Std.2 | 200 μL of Std.1 (30 μg/mL) | 200 | 15 |
| Std.3 | 200 μL of Std.2 (15 μg/mL) | 200 | 7.5 |
| Std.4 | 200 μL of Std.3 (7.5 μg/mL) | 200 | 3.75 |
| Std.5 | 200 μL of Std.4 (3.75 μg/mL) | 200 | 1.875 |
| Std.6 | 200 μL of Std.5 (1.875 μg/mL) | 200 | 0.938 |
| Std.7 | 200 μL of Std.6 (0.938 μg/mL) | 200 | 0.469 |
| Std.8 | 200 μL of Std.7 (0.469 μg/mL) | 200 | 0.234 |
注意：每次实验都要做一次标准品检测，制作标曲；稀释后的标准品溶液不稳定，必须在4小时内使用。
3. 样本制备
注意：建议使用新鲜样本。如果不立即使用，可将样品在-80℃下保存一个月。测定时，应控制解冻的温度和时间。室温环境下解冻时，需在4h内完成样品解冻。
3.1 动物组织
称取约0.2 g样品于玻璃管，加入2 mL Tissue Extraction Buffer，置于110℃烘箱，水解6-12 h，冷却后用氢氧化钠溶液调节pH为7.0左右，用去离子水定容至4 mL，12,000g，25℃，离心20 min，取上清待测。
3.2 细菌和细胞
收集500万细菌或细胞到离心管内，加入1 mL Cell Extraction Buffer，于高压消毒器中15磅保持30 min，自然降压后用浓盐酸调节pH为7.0左右，用去离子水定容至2 mL，12,000g，25℃，离心20 min，取上清待测。
3.3 血清中游离HYP提取
取0.1 mL血清，加入0.5 mL无水乙醇使蛋白质沉淀，8,000g，4℃离心5 min。将上清倒入另一个EP管，氮吹或沸水浴将乙醇挥发干。冷却后再加入0.2 mL 50%异丙醇，充分溶解混匀待测。
注意：
(1)如需测定蛋白浓度，推荐使用阿拉丁B665595 BCA蛋白定量试剂盒或R1491648 即用型BCA蛋白定量试剂盒。
(2)提取过程中可能有黑色物质生成，若长时间不能消化，可能为碳化的物质，不影响实验。
4. 实验步骤
4.1 酶标仪或可见光分光光度计预热30 min以上，调节波长到560 nm，可见光分光光度计用去离子水调零。
4.2 操作表（下述操作在1.5 mL离心管中操作）
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4.3 充分混匀，60℃反应20 min，取出后室温静置15 min，取200 μL至微量玻璃比色皿或96孔板中，记录560 nm处吸光值，空白孔记为A空白，标准孔记为A标准，测定孔记为A测定。计算ΔA测定=A测定-A空白，ΔA标准=A标准-A空白。
注意：空白管和标准管只需做1-2次。
实验之前建议选择2-3个预期差异大的样本做预实验。如果ΔA测定小于0.005可适当加大样本量。如果ΔA测定大于30 μg/mL的ΔA标准，样本可用相应提取液一步稀释，计算结果乘以稀释倍数，或减少提取用样本量。
5. 结果计算
注意：我们为您提供的计算公式，包括推导过程计算公式和简洁计算公式。两者完全相等。建议以加粗的简洁计算公式为最终计算公式。
5.1 标准曲线的绘制
以标准溶液浓度为x轴，ΔA标准为y轴，绘制标准曲线，得到标准方程，将ΔA测定带入方程得到x (µg/mL)。
5.2 HYP含量的计算
(1) 按样本蛋白浓度计算
HYP (µg/mg prot) = x × V样 ÷ (Cpr × V样) = x ÷ Cpr
(2) 按样本鲜重计算
HYP (µg/g 鲜重) = x × V反总 ÷ (V样 ÷ V组总 × W) = 20x ÷ W
(3) 按细菌或细胞数量计算
HYP (µg/104) = x × V反总 ÷ (V样 ÷ V胞总 × n) = 10x ÷ n
(4）按样本体积计算
HYP (µg/mL) = x × V反总 ÷ (V样本 ÷ 2) = 10x
参数说明：
x：反应总体积，0.3 mL
V样：反应中样品体积，0.06 mL
V组总：加入组织的提取液体积，4 mL
V胞总：加入细菌或细胞的提取液体积，2 mL
Cpr：样本蛋白浓度，mg/mL
W：样本重量，g
n：细菌或细胞数量，万
2：血清吹干后复溶的倍数
注意事项
1. 正式检测前，建议选择2-3个预期差异较大的样本进行预实验。
2. 对于组织样本及细胞样本等，可通过测定蛋白浓度来进行样本间结果均一化。推荐使用阿拉丁B665595 BCA蛋白定量试剂盒或R1491648 即用型BCA蛋白定量试剂盒。
3. 生化检测试剂普遍具有刺激性、生物毒性等，为了您的安全和健康，请在实验全程做好生物安全防护，穿戴实验服、口罩、手套、头套等安全护具，在通风橱、生物安全柜中进行实验。
4. 本产品仅供科学研究使用，不适用于临床诊断。
Hydroxyproline (HYP) is a non-essential amino acid and one of the major components of collagen tissue in vivo. Except for a small amount contained in elastin, most hydroxyproline exists in collagen. The content of hydroxyproline can serve as a critical indicator for evaluating collagen tissue metabolism in the body and is of great significance for determining the degree of tissue fibrosis.
Detection Principle
Free hydroxyproline is released from the test sample via acid hydrolysis and oxidized into pyrrole by Chloramine-T. Pyrrole further reacts with p-Dimethylaminobenzaldehyde to form a red-colored compound, which has a characteristic absorption peak at 560 nm. The content of hydroxyproline can be quantitatively determined according to the change in absorbance value.
Applicable Samples
Animal tissues, bacteria and cultured cells, serum, plasma and other samples
Note: It is recommended to select 2-3 samples with significant expected differences for pre-experiment before formal detection.
Reagents, consumables and Equipments not provided
Microplate reader or visible light spectrophotometer (capable of measuring absorbance at 560 nm)
Water bath, centrifuge, oven
96-well plate or micro glass cuvette, adjustable pipette and tips, 1.5 mL centrifuge tubes, glass tubes
Deionized water, concentrated hydrochloric acid, anhydrous ethanol, isopropanol, sodium hydroxide
Operating Procedure
1. Reagent Preparation
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2. Standard Working Solution Preparation
Dilute the 0.5 mg/mL L-Hydroxyproline standard according to the table below to prepare gradient standard solutions:
No. | Volume of Stock Standard | Volume of Deionized Water | Final Concentration (μg/mL) |
Std.1 | 24 μL 0.5 mg/mL Standard | 376 μL | 30 |
Std.2 | 200 μL of Std.1 (30 μg/mL) | 200 μL | 15 |
Std.3 | 200 μL of Std.2 (15 μg/mL) | 200 μL | 7.5 |
Std.4 | 200 μL of Std.3 (7.5 μg/mL) | 200 μL | 3.75 |
Std.5 | 200 μL of Std.4 (3.75 μg/mL) | 200 μL | 1.875 |
Std.6 | 200 μL of Std.5 (1.875 μg/mL) | 200 μL | 0.938 |
Std.7 | 200 μL of Std.6 (0.938 μg/mL) | 200 μL | 0.469 |
Std.8 | 200 μL of Std.7 (0.234 μg/mL) | 200 μL | 0.234 |
Note: Prepare a standard curve with standard solutions in each independent experiment. The diluted standard solutions are unstable and must be used within 4 hours.
3. Sample Preparation
Note: Fresh samples are recommended for detection. Samples can be stored at -80 °C for up to one month if not used immediately. Control the thawing temperature and time during detection; complete thawing within 4 hours at room temperature.
3.1 Animal Tissue Samples
Weigh approximately 0.2 g tissue into a glass test tube, add 2 mL Tissue Extraction Buffer. Hydrolyze in a drying oven at 110 °C for 6-12 hours. After cooling, adjust the pH to around 7.0 with sodium hydroxide solution, and dilute to 4 mL with deionized water. Centrifuge at 12,000 g, 25 °C for 20 minutes, and collect the supernatant for testing.
3.2 Bacteria & Cultured Cell Samples
Collect 5 million bacteria or cells into a centrifuge tube, add 1 mL Cell Extraction Buffer. Treat in an autoclave at 15 psi for 30 minutes, then cool naturally. Adjust the pH to around 7.0 with concentrated hydrochloric acid, dilute to 2 mL with deionized water. Centrifuge at 12,000 g, 25 °C for 20 minutes, and collect the supernatant for testing.
3.3 Extraction of Free HYP in Serum
Add 0.5 mL absolute ethanol to 0.1 mL serum for protein precipitation. Centrifuge at 8,000 g, 4 °C for 5 minutes. Transfer the supernatant to a new EP tube, evaporate ethanol completely by nitrogen blowing or water bath heating. After cooling, add 0.2 mL 50% isopropanol to fully dissolve the residue for testing.
Precautions for Sample Preparation
4. Experimental Operation Steps
4.1 Preheat the microplate reader or visible spectrophotometer for more than 30 minutes, set the wavelength to 560 nm, and zero the spectrophotometer with deionized water. 4.2 Reaction System Setup (Operate in 1.5 mL centrifuge tubes)
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4.3 Mix all tubes thoroughly, incubate at 60 °C for 20 minutes, then cool at room temperature for 15 minutes. Transfer 200 μL of each reaction solution into a 96-well plate or micro cuvette, and measure the absorbance at 560 nm. Record ABlank, AStandard and ASample. Calculate ΔASample = ASample - ABlank, ΔAStandard = AStandard - ABlank. Note: The blank and standard groups only need to be repeated 1-2 times per experiment.Perform pre-experiments with 2-3 samples of large content differences before formal testing. If ΔASample is lower than 0.005, increase the sample dosage appropriately. If ΔASample exceeds the absorbance of 30 μg/mL standard solution, dilute the sample with the corresponding extraction buffer and multiply the dilution factor in final calculation, or reduce the initial sample weight/volume.
5. Result Calculation
Note: Two formulas are provided, including the derived formula and the simplified formula. Both formulas are completely equivalent. The bold simplified formula is recommended for final calculation.
5.1 Standard Curve
Plotting Plot the standard curve with the standard solution concentration on the x-axis and ΔAstandard on the y-axis to obtain the standard equation. Substitute ΔAsample into the equation to calculate the value of x (µg/mL).
5.2 Calculation of HYP Content
(1) Calculation based on sample protein concentration
HYP (µg/mg prot) = x × Vsample ÷ (Cpr × Vsample) = x ÷ Cpr
(2) Calculation based on sample fresh weight
HYP (µg/g fresh weight) = x × Vtotal reaction ÷ (Vsample ÷ Vtotal tissue extract × W) = 20x ÷ W
(3) Calculation based on bacterial or cell count
HYP (µg/104 cells) = x × Vtotal reaction ÷ (Vsample ÷ Vtotal cell extract × n) = 10x ÷ n
(4) Calculation based on sample volume
HYP (µg/mL) = x × Vtotal reaction ÷ (Vsample ÷ 2) = 10x
Parameter Explanation
x: The calculated concentration from standard curve (μg/mL)
Vtotal reaction: Total reaction volume, 0.3 mL
Vsample added: Sample volume added to reaction system, 0.06 mL
Vtissue extraction total: Total extraction buffer volume for tissue samples, 4 mL
Vcell extraction total: Total extraction buffer volume for cell/bacteria samples, 2 mL
Cpr: Protein concentration of sample (mg/mL)
W: Fresh weight of tissue sample (g)
n: Number of bacteria or cells (10⁴ units)
2: Redissolution dilution factor for serum sample
Precautions
Pre-experiment with 2-3 differential samples is required before formal detection.
Normalize experimental results among different samples by determining protein concentration for tissue and cell samples. Aladdin B665595 or R1491648 BCA protein assay kits are recommended.
Biochemical reagents are irritant and biologically toxic. Wear lab coat, gloves, mask and hair cover throughout the experiment. All operations must be performed in a fume hood or biosafety cabinet for personal protection.
This product is for scientific research only and is not intended for clinical diagnosis.
H1515817 | Component | 96T | Storage |
H1515817A | Cell Extraction Buffer | 120 mL | 2-8℃. |
H1515817B | 试剂一 | 8.4 mL | 2-8℃. Store in the dark. |
H1515817C | 试剂二 | 8.4 mL | 2-8℃. Store in the dark. |
H1515817D | 标准品 | 0.5 mL | 2-8℃. Store in the dark. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0939492 |
分析证书 | H1515817 |
| ZJ26F0434543 |
分析证书 | H1515817 |
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