솔라바이오 하이드록시프롤린 (우울) 활동 분석 키트

하이드록시프롤린 (우울) 활동 분석 키트

메모: 테스트 전에 예측을 위해 2~3개의 서로 다른 샘플을 채취합니다..

탐지장비: 분광 광도계/마이크로플레이트 리더

고양이 번호: BC0255

크기: 100T/96S

구성요소:

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

시약 I: 8 밀리리터×1, store at 4℃ and protect from light. 시약 II: 8 밀리리터×1, store at 4℃ and protect from light. 기준: 0.5 밀리리터×1, 0.5 mg/mL hydroxyproline, 4℃에 보관.

설명:

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. 그러므로, 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.

필수이지만 제공되지 않음:

저울, oven, glass tube, 원심분리기, 욕조, spectrophotometer/microplate reader, micro glass cuvette/96-well plate, 6 mol/L HCl and distilled water.

절차:

나. 샘플 준비

1. 1. 조직 샘플:

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

식힌 후, 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 rpm 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. 세포:

가져가다 5 백만개의 세포, 추가하다 1 mL의 추출 용액, boil, or oven at 110℃ for 2 에게 6 hours to digest to transparent state.

식힌 후, 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 rpm 20 minutes at 25℃ (if there is still impurity after centrifugation, it can be removed by filtration).

Take the supernatant for test.

II. Detection

1. 예열 분광 광도계/마이크로플레이트 판독기 30 분, 파장을 조정하다 560 nm and set zero with distilled
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.

잘 섞다, incubate at 60℃ for 20 분, leave it at room temperature for 15 분, 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. 계산

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 밀리리터;

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

여: Fresh weight of sample, g;

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

심폐소생술: Concentration of sample protein, mg/mL.

메모

1. OD 값이 다음보다 큰 경우 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.

실험예

• 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 우물 접시, 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.

최근 제품 인용

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

관련 상품

BC1550/BC1555 Glutamic-pyruvic Transaminase (GPT) Activity Assay Kit BC1560/BC1565 Glutamic-oxalacetic Transaminase (GOT) Activity Assay Kit BC0290/BC0295 Proline (PRO) 콘텐츠 분석 키트

BC1570/BC1575 Amino Acid (AA) Content Assay Kit BC0180/BC0185 Cysteine (Cys) Content Assay Kit BC1580/BC1585 Glutamic Acid (Glu) 콘텐츠 분석 키트

기술 사양:

Detection Limit: 0.057 μg/mL

선형 범위: 0.117-30 μg/mL