Solarbio Hidroxiprolina (HYP) Kit de ensayo de actividad

Hidroxiprolina (HYP) Kit de ensayo de actividad

Nota: Tome dos o tres muestras diferentes para predecir antes de la prueba..

Detection equipment: Spectrophotometer/Microplate reader

No gato: BC0255

Tamaño: 100T/96S

Componentes:

Extraer solución: 6 mol/L hydrochloric acid (HCl), self-provided reagent. Concentrated HCl（ 37%） : H2O(V/V) =1:1, stored at room temperature.

reactivo yo: 8 mL×1, store at 4℃ and protect from light. Reactivo II: 8 mL×1, store at 4℃ and protect from light. Estándar: 0.5 mL×1, 0.5 mg/mL hydroxyproline, store at 4℃.

Descripción:

HYP is one of the main components of collagen in the body. Most of the collagen is distributed in the skin, tendon, cartilage, and blood vessels et al. Por lo tanto, the content of HYP is an important index reflecting the metabolism and fibrosis degree of collagen tissue.

The sample is hydrolyzed to produce free HYP, which is further oxidized by chloramine T. The oxidized product reacted with p-Dimethylaminobenzaldehyde to produce a red compound with characteristic absorption peak at 560 Nuevo Méjico. The content of HYP can be calculated by measuring the absorption value of sample hydrolysate at 560 Nuevo Méjico.

Required but not provided:

Scales, oven, glass tube, centrífugo, baño de agua, spectrophotometer/microplate reader, micro glass cuvette/96-well plate, 6 mol/L HCl and distilled water.

Procedimiento:

I. preparación de la muestra

1. 1. Muestra de tejido:

Weigh about 0.2 g of the sample into the glass tube, cut the tissue into pieces as much as possible for digestion. Agregar 2 mL of Extract solution and the cover is slightly loose and not airtight, boil it or bake it in 110℃ oven for 2 a 6 hours to digest it until there is no visible big lump.

Después de enfriar, adjust the pH to 6-8 con 10 mol/L NaOH (Do not over acid or over alkali) then constant volume to 4 mL with distilled water. Centrifugation at 16000 rpm para 20 minutes at 25℃ (if there is still impurity after centrifugation, it can be removed by filtration).

Take the supernatant for test. (Black substance may be formed in the process, and if it cannot be digested for a long time, it may be carbonized substance. It does not affect the experiment).

2. Células:

Llevar 5 millones de células, agregar 1 mL de solución de extracto, boil, or oven at 110℃ for 2 a 6 hours to digest to transparent state.

Después de enfriar, adjust the pH to 6-8 con 10 mol/L NaOH (Do not over acid or over alkali) then constant volume to 2 mL with distilled water. Centrifugation at 16000 rpm para 20 minutes at 25℃ (if there is still impurity after centrifugation, it can be removed by filtration).

Take the supernatant for test.

II. Detección

1. Precaliente el espectrofotómetro/lector de microplacas para 30 minutos, ajustar la longitud de onda a 560 nm y poner a cero con destilado
2. Dilute the standard solution to 30, 15, 7.5, 3.75, 1.875, 0.938, 0.469, 0.234 µg/mL.
3. Add reagents as the following table.

Mezclar bien, incubate at 60℃ for 20 minutos, leave it at room temperature for 15 minutos, take 200μL of the reaction solution into micro glass cuvette/96 well flat-bottom plate and test the absorbance value of at 560 Nuevo Méjico. ΔA = AT – AB.

III. Cálculo

1. Making of standard

When making the standard curve, the concentration of the standard solution is taken as the x-axis, and the ΔAS (ΔAS =AS-AB) is taken as the y-axis. The linear equation y=kx+b is obtained. Take ΔAT (AT-AB) to the equation to acquire x.

2. Calculation of hydroxyproline content:calculated according to the fresh weight of the sample:

Tissue hydroxyproline content (μg/g fresh weight) = x×VS÷(W×VS÷VTE) = 4x÷W.

• Calculated according to the sample protein concentration:

Tissue hydroxyproline content (μg/mg prot) = x×VS÷(Cpr×VS) = x÷Cpr.

• Calculated according to the number of bacteria orcells:

Cell hydroxyproline content (μg/104 cell) = x×VS÷(N×VS÷VCE )= 2x÷N.

VS: Volume of added sample, 0.06 mililitros;

VTE: Volume of tissue extract solution, 4 mililitros; VCE: Volume of cell extract solution, 2 mililitros;

W.: Fresh weight of sample, gramo;

norte: Number of cells,104 as units, 10000;

RCP: Concentration of sample protein, mg/mL.

Nota

1. If the OD value is greater than 1.0, the sample should be diluted properly and then determined. Pay attention to multiply the dilution multiple in the calculation formula.
2. The reagent has certain toxicity. Please take protective measures during operation to prevent inhalation or contact with skin.
3. When calculating according to the sample protein concentration, the protein in the sample itself needs to be extracted separately and determined.

Experimental examples

• 0.2g of mouse leg is taken for sample treatment, and the supernatant is taken out and operated according to the determination steps. ΔA =AT-AB = 0.177-0.06 = 0.117 is measured by 96 buen plato, and the standard curve y = 0.0383x + 0.022 is brought in, and x = 2.48 is

The content of hydroxyproline in tissue (μg/g mass) = 4x ÷ W = 4 × 2.48 ÷ 0.2 = 49.6 μg/g mass.

Recent Product Citations

• Litong Fan,Jiaqing Chen,Yanmeng Tao,et al. Enhancement of the chondrogenic differentiation of mesenchymal stem cells and cartilage repair by ghrelin. Journal of Orthopaedic Research. January 2019;(IF3.043)

Productos relacionados

BC1550/BC1555 Glutamic-pyruvic Transaminase (GPT) Activity Assay Kit BC1560/BC1565 Glutamic-oxalacetic Transaminase (GOT) Activity Assay Kit BC0290/BC0295 Proline (PRO) Kit de ensayo de contenido

BC1570/BC1575 Amino Acid (AA) Content Assay Kit BC0180/BC0185 Cysteine (Cys) Content Assay Kit BC1580/BC1585 Glutamic Acid (Glu) Kit de ensayo de contenido

Especificaciones técnicas:

Detection Limit: 0.057 µg/mL

Rango lineal: 0.117-30 µg/mL