Solarbio 산화 글루타티온 (GSSG) 콘텐츠 분석 키트

산화된 글루타티온 (GSSG) 콘텐츠 분석 키트

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

운영장비: 분광 광도계/마이크로플레이트 리더

카탈로그 번호: BC1185

크기:100T/96S

구성요소:

시약 I:100 밀리리터×1. 4℃에서 보관. 시약 II: 130 μL×1. 4℃에서 보관. 시약 III: 20 밀리리터×1. 4℃에서 보관. 시약 IV:2.5 밀리리터×1. 4℃에서 보관.

시약 V: Powder ×1. 4℃에서 보관. Dissolve with 2.5 용액을 사용할 경우 증류수 mL, then split into smaller packages, -20℃에 보관.

시약 VI:12.5 μL×1. 4℃에서 보관. Prepare Reagent VI and distilled water according to the sample size at the ratio of 1:20 (V: V) 사용하기 전에.

기준: 가루 10 mg×1. 4℃에서 보관.

제품 설명

산화된 글루타티온(GSSG) is an oxidized form of glutathione (GSH), also known as dithioneglutathione, formed by the oxidation of two molecules of glutathione. GSSG is reduced to GSH by glutathione reductase, so most of the body is in the reduced form. The determination of GSH and GSSG content and ratio of GSH/GSSG in cells can reflect the redox status of cells. This kit utilizes reaction of glutathione and 5, 5′-dithiobis-2-nitrobenoic acid (DTNB) to produce 5-thio-2- nitrobenzoic acid. 5-thio-2- nitrobenzoic acid has the largest absorption at wavelength of 412 nm, and 2-Vinylpyridine inhibit reduced glutathione in the original of samples, and then using glutathione reductase to reduce GSSG to GSH, determining the content of Oxidized Glutathione.

기술 사양

Minimum Detection Limit：3.211 μg/mL

선형 범위：3.9-125 μg/mL

필요하지만 제공되지 않는 시약 및 장비.

Analytical balance, 모르타르/균질화기, refrigerated centrifuge, 욕조, 조절 가능한 피펫, spectrophotometer/ microplate reader, 마이크로 유리 큐벳/96웰 평면 바닥 플레이트.

절차

나. 견본 준비

1. 1. 조직 샘플

Wash fresh tissues with PBS for twice, 그런 다음 추가 0.1 g of sample into the homogenizer (the homogenizer has been rinsed with Reagent I and placed on ice before use). 추가하다 1 mL의 시약 I (the proportion of tissue and reagents can be kept constant), 얼음 위에서 완전히 분쇄 (using liquid nitrogen will have a better grinding effect). 원심분리기 8000 ×g and 4℃ for 10 분, take the supernatant and place it at 4℃ for test. (The supernatant can be stored at -80℃ for 10 days.)

2. 혈액 샘플

혈장: Sample is centrifuged at 600 ×g and 4℃ for 10 분. Absorbing the upper plasma into another tube add with same volume Reagent I. 원심분리기 8000 ×g and 4℃ for 10 분, take the supernatant and place it at 4℃ for test. (The Supernatant can be stored at -80℃ for 10 days.)

Blood cell:Sample is centrifuged at 600 ×g and 4℃ for 10 분. Discarding the upper plasma, wash with treble volume of PBS for 3 타임스 (mix blood cell with PBS centrifuge at 600 ×g 10 분), add equal volume of Reagent I. After mixing, it is placed at 4℃ for 10 분. 원심분리기 8000 ×g 10 분, take the supernatant and place it at 4℃ for test. (The supernatant can be stored at -80℃ for 10 days.)

3. 세포 샘플

Harvesting cell should not less than 106,then wash with PBS for twice (mix cell with PBS centrifuge at 600×g for 10 분), mix precipitated cell with the volume of PBS for 3 타임스. Repeated freezing and thawing 2–3 times (suggest frozen in liquid nitrogen, dissolved in 37℃ water bath). 원심분리기 8000 ×g 10 분, take the supernatant and place it at 4℃ for test. (The supernatant can be stored at -80℃ for 10 days.)

II. 절차

1. 예열 분광 광도계/마이크로플레이트 판독기 30 분, 파장을 조정하여 412 nm, 증류수로 카운터를 0으로 설정
2. Preheat Reagent II in water bath for 30 분: 37℃ (mammal cell) 또는 25℃ (다른 종).
3. The standard dilution: dissolve standard with 1 증류수 1mL (4℃) to a concentration of 10 mg/mL. Take suitable solution to prepare the standard of concentration of 125μg/mL、 5μg/mL、31.25μg/mL、15.625μg/mL、7.8125μg/mL、3.90625μg/mLand0μg/mL (The diluent is a ten-fold diluted Reagent I).

4. 추가하다 20 μL of diluted standard or sample to 0.5 mL centrifuge tube, 추가하다 1 시약 II의 μL, incubate at 37 ℃ for 30 minutes after mixing.
5. Make standard curves

After the incubation, 추가하다 140 μL of Reagent III, 20 시약 IV의 μL, 20μL of Reagent V, 그리고 2 μL of Reagent VI to the standard tube in sequence. After rapid mixing, the light absorption A1 and A2 of 30 s and 150 s respectively were measured at 412 nm. Absorbance (A2-A1) is the abscissa (엑스) and concentration is the ordinate (와이), making the standard curve.

추가하다 140 μL of Reagent III, 20 시약 IV의 μL, 20 μL of Reagent V, 그리고 2 μLof Reagent VI to the sample tubes in sequence. After rapid mixing, the light absorption A1 and A2 of 30 s and 150 s respectively were measured at 412 nm, ΔA= A2- A1.

III. 계산

According to the standard curve, sample ΔA into the formula (엑스), calculate the sample concentration of y

(μg/ml).

• 단백질 농도

GSSH (μg / mg prot)=y×Vrv÷Vrv÷Cpr =y÷Cpr

• 샘플 중량

GSSH (μg /g)= y×Vrv÷(Vrv÷Vsv×W)= y÷W

• 셀라마운트

GSSH (μg /106 셀)= y×Vrv÷(Vrv÷Vsv×N)= y÷N

• Solution volume

GSSH (μg / 밀리리터)= y×Vrv/Vs= 2y

N: 셀량,106；

로프: 총 반응량, 0.203 밀리리터；

VSV: The volume of supernatant was added into the reaction system, 20 μL=0.02 mL； 여: 샘플 중량, g;

심폐소생술: 상층액 단백질 농도, mg/mL.

노트:

1. The sample needs to be homogenized completely. If the test cannot be completed temporarily, it can be stored at-80℃.
2. If the content of GSSG content in the sample is uncertain, several gradients can be diluted before measurement.
3. This method uses the enzymatic reaction rate to calculate the substrate concentration and complete readings as accurately as possible at 30 그리고 150
4. Reagent I contained protein precipitant, the supernatant could not be used for protein concentration determination. If the protein content needs to be determined, take another tissue.

참조:

• Alpert A J, Gilbert H F. Detection of oxidized and reduced glutathione with a recycling postcolumn reaction[제이]. Analytical biochemistry, 1985, 144(2):553-562.
• Owens C W I, Belcher R A colorimetric micro-method for the determination of glutathione[제이]. Biochemical Journal, 1965, 94(3): 705.

관련 상품：

BC1170/ BC1175 Reduced Glutathione（GSH）Assay Kit

BC1190/ BC1195 Glutathione Peroxidase Assay Kit

BC0350/ BC0355 Glutathione S-transferase(GST) 활동 분석 키트

BC1160/ BC1165 Glutathione Reductase (GR) 분석 키트