Solarbio Hydroxyprolin (HYP) Aktivitätstest-Kit

Hydroxyprolin (HYP) Aktivitätstest-Kit

Notiz: Nehmen Sie vor dem Test zwei oder drei verschiedene Proben zur Vorhersage.

Erkennungsausrüstung: Spektrophotometer/Mikroplattenleser

Katze nein: BC0255

Größe: 100T/96S

Komponenten:

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

Reagenz I: 8 ml×1, store at 4℃ and protect from light. Reagenz II: 8 ml×1, store at 4℃ and protect from light. Standard: 0.5 ml×1, 0.5 mg/mL hydroxyproline, Speichern Sie bei 4 ℃.

Beschreibung:

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. daher, 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 nm. The content of HYP can be calculated by measuring the absorption value of sample hydrolysate at 560 nm.

Erforderlich, aber nicht bereitgestellt:

Scales, oven, glass tube, Zentrifuge, Wasserbad, Spektrophotometer/Mikroplattenleser, micro glass cuvette/96-well plate, 6 mol/L HCl and distilled water.

Verfahren:

ICH. Probenvorbereitung

1. 1. Gewebeprobe:

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

Nach dem Abkühlen, adjust the pH to 6-8 with 10 mol/L NaOH (Do not over acid or over alkali) then constant volume to 4 mL with distilled water. Centrifugation at 16000 U/min für 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. Zellen:

Nehmen 5 Millionen Zellen, hinzufügen 1 ml Extraktlösung, boil, or oven at 110℃ for 2 Zu 6 hours to digest to transparent state.

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

Take the supernatant for test.

II. Erkennung

1. Vorheizen -Spektrophotometer/Mikroplattenleser für 30 Protokoll, Wellenlänge anpassen 560 nm und mit destilliertes Null einstellen
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.

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

III. Berechnung

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 ml;

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

W: Fresh weight of sample, G;

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

Cpr: Concentration of sample protein, mg/ml.

Notiz

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.

Experimentelle Beispiele

• 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 Brunnenplatte, 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.

Neuere Produktzitate

• 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)

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Technische Spezifikationen:

Detection Limit: 0.057 μg/ml

Linearer Bereich: 0.117-30 μg/ml