Hydroxyproline (HYP) Content Assay Kit (Ehrlich, Micro Method)

Cat. No.: H1515817

GRADE & PURITY BioReagent

Overview

  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

Reagent Name	Notes	Storage
Tissue Extraction Buffer (Self-provided)	Ready-to-use; 6 mol/L hydrochloric acid, concentrated hydrochloric acid: deionized water (V/V)=1:1	Store at room temperature
Cell Extraction Buffer	Ready-to-use; equilibrate to room temperature before use	Store at 4°C
Reagent I	Ready-to-use; equilibrate to room temperature before use	Store at 4°C, protect from light
Reagent II	Ready-to-use; equilibrate to room temperature before use	Store at 4°C, protect from light
Standard	Ready-to-use; 0.5 mg/mL L-hydroxyproline standard; equilibrate to room temperature before use	Store at 4°C, protect from light

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

• For protein concentration determination, Aladdin BCA Protein Assay Kit (Cat.No. B665595) or Ready-to-use BCA Protein Assay Kit (Cat.No. R1491648) is recommended.
• Black substances may be generated during extraction; undigested carbonized substances will not interfere with the experimental results.

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)

Reagent	Blank Tube (μL)	Standard Tube (μL)	Sample Tube (μL)
Sample Solution	0	0	60
Standard Solution	0	60	0
Reagent I	60	60	60

Mix thoroughly and incubate at room temperature for 20 minutes.

Reagent II	60	60	60
Deionized Water	180	120	120

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 A_Blank, A_Standard and A_Sample. Calculate ΔA_Sample = A_Sample - A_Blank, ΔA_Standard = A_Standard - A_Blank. 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 ΔA_Sample is lower than 0.005, increase the sample dosage appropriately. If ΔA_Sample 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 ΔA_standard on the y-axis to obtain the standard equation. Substitute ΔA_sample 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 × V_sample ÷ (C_pr × V_sample) = x ÷ C_pr 

(2) Calculation based on sample fresh weight 

HYP (µg/g fresh weight) = x × V_{total reaction} ÷ (V_sample ÷ V_{total tissue extract} × W) = 20x ÷ W 

(3) Calculation based on bacterial or cell count 

HYP (µg/10⁴ cells) = x × V_{total reaction} ÷ (V_sample ÷ V_{total cell extract} × n) = 10x ÷ n 

(4) Calculation based on sample volume 

HYP (µg/mL) = x × V_{total reaction} ÷ (V_sample ÷ 2) = 10x 

Parameter Explanation 

x: The calculated concentration from standard curve (μg/mL) 

V_{total reaction}: Total reaction volume, 0.3 mL 

V_{sample added}: Sample volume added to reaction system, 0.06 mL 

V_{tissue extraction total}: Total extraction buffer volume for tissue samples, 4 mL 

V_{cell extraction total}: Total extraction buffer volume for cell/bacteria samples, 2 mL 

C_pr: 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

1. Pre-experiment with 2-3 differential samples is required before formal detection.

2. 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.

3. 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.

4. This product is for scientific research only and is not intended for clinical diagnosis.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Stability And Storage

Store at 2-8℃ long term (6 months). Store in the dark.

Contents & Storage

H1515817	Component	96T	Storage
H1515817A	Cell Extraction Buffer	120 mL	2-8℃.
H1515817B	Reagent I	8.4 mL	2-8℃. Store in the dark.
H1515817C	Reagent II	8.4 mL	2-8℃. Store in the dark.
H1515817D	Standard	0.5 mL	2-8℃. Store in the dark.