Solarbio 환원 글루타티온 (GSH) 콘텐츠 분석 키트

Reduced Glutathione (GSH) 콘텐츠 분석 키트

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

운영장비: Spectrophotometer/Microplate Reader

카탈로그 번호: BC1175

크기: 100T/96S

구성요소:

시약 I: 100 밀리리터×1. 4℃에서 보관.

시약 II: 20 밀리리터×1. 4℃에서 보관.

시약 III: 8 밀리리터×1. 4℃에서 보관, protect from light.

기준: 가루 10 mg×1. 4℃에서 보관, protect from light.

제품 설명

Glutathione is a natural tripeptide composed of glutamic acid (Glu), cysteine (Cys) and glycine (Gly). It is a kind of compound containing sulfhydryl group (-SH), which widely exists in animal tissue, plant tissue, microorganism, and yeast. Glutathione can react with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) to produce 2-nitro-5-mercaptobenzoic acid and glutathione disulfide (GSSG). 2-nitro-5-mercaptobenzoic acid is a yellow product, with the maximum absorption at 412 nm.

기술 사양

Minimum Detection Limit：3.763 μg/mL

선형 범위：12.5-400 μg/mL

필요하지만 제공되지 않는 시약 및 장비

Analytical balance, 모르타르/균질화기, 저온 원심분리기, 욕조, 조절 가능한 피펫, spectrophotometer/microplate reader, micro glass cuvette or 96 well flat-bottom plate and distilled water.

절차

나. 샘플 준비

1. 조직 샘플

Wash fresh tissues with PBS for twice, 그런 다음 추가 0.1 g of animal/plant tissue into 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 10 4℃에서 분, take the supernatant and place it at 4℃ for test. (If the test cannot be completed temporarily, the supernatant can be stored at -80℃ for 10 days.)

2. 혈액 샘플

혈장: Sample is centrifuged at 600 ×g 10 4℃에서 분. Absorbing the upper plasma into another tube with adding the same volume Reagent I. 원심분리기 8000 ×g 10 4℃에서 분, take the supernatant and place it at 4℃for test. (If the test cannot be completed temporarily, the supernatant can be stored at -80℃ for 10 days.)

Blood cell: Sample is centrifuged at 600 ×g 10 4℃에서 분. Discarding the upper plasma, wash with three times volume of PBS for 3 타임스 (re-suspend blood cell with PBS, 원심분리기 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. (If the test cannot be completed temporarily, the supernatant can be stored at -80℃ for 10 days.)

3. Cell 견본

Harvesting cell should not less than 106,then wash it with PBS for twice (re-suspend cell with PBS, 원심분리기 600 ×g 10 분). The volume of Reagent I added is three times the volume of cell precipitation to re-suspend the cells. Repeated freezing and thawing 2–3 times (It is suggested that frozen in liquid nitrogen, dissolved in 37℃ water bath). 원심분리기 8000 ×g 10 분, take the supernatant and place it at 4℃ for test. (If the test cannot be completed temporarily, 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 0을 설정하다
2. Preheat Reagent II in 37℃ (mammal cell) 또는 25℃ (다른 종) 수욕 30
3. Blank tube determination: take micro glass cuvette, 추가하다 20 증류수 μL, 140 시약 II의 μL, 40 μL of Reagent III in turn, 잘 섞다, place for 2 분, and measure 412 nm absorbance AB.
4. Making standard curve

달다 1 mg of standard and dissolve it with 1 mL of distilled water to obtain the concentration of 1 mg/mL. Take the appropriate solution to prepare the standards with the concentration of 200 μg/mL, 100 μg/mL, 50 μg/mL, 25 μg/mL and 12.5 μg/mL (Dilute Reagent I ten times before diluting the standard solution).

Take a 1.5 mL EP tube and add 20 μL of standard, 140 μL of Reagent II and 40 μL of Reagent III in turn. After each tube is evenly mixed, it is allowed to stand for 2 분. 에서 흡광도를 측정합니다. 412 nm, and absorbance minus AB as abscissa. Make the standard curve according to the absorbance (엑스) and concentration (y, μg/mL).

5. Sample tube test: take micro glass cuvette, 추가하다 20 샘플의 μL, 140 시약 II의 μL, 40 μL of Reagent III in turn, 잘 섞다, and then stand for 2 minutes to test the absorbance ATat 412 nm, ΔA = AT – AB.
6. The operation of the microplate reader is the same as that of the spectrophotometer, and the operation is as fast as

III. 계산

According to the standard curve, take sample ΔA into the formula(엑스), and calculate the sample concentration y (μg/mL).

• 단백질 농도

GSH (μg/mg prot)=y×VRV÷VRV÷Cpr =y÷Cpr

• 샘플 중량

GSH (μg/g)=y×VRV÷(VRV÷VSV×W)= y÷W

• 셀량

GSH (μg/104cell)=y×VRV÷(VRV÷VSV×N)= y÷N

• Solution volume GSH (μg/mL)=2y

N: 셀량, 106;

VSV: 총 상층액 부피, 1 밀리리터；

VRV: Supernatant volume added into the reaction system, 20 μL=0.02 mL； 여: 샘플 중량, g;

심폐소생술: 상층액 단백질 농도, mg/mL.

2: The volume of plasma (blood cells) is diluted by one time.

메모:

1. The sample needs to be homogenized completely. If the test cannot be completed temporarily, it can be stored at-80℃.
2. 기준: Reduced glutathione is prepared when the solution will be
3. If the GSH content in the sample is uncertain, Dilute the sample for several gradients before
4. Because Reagent I contain protein precipitant, the supernatant cannot 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 V. A colorimetric micro-method for the determination of glutathione[제이]. Biochemical Journal, 1965, 94(3):

관련 상품：

BC1180/ BC1185 Oxidized Glutathione（GSSG）Assay Kit

BC1190/ BC1195 Glutathione Peroxidase Assay Kit

BC1150/ BC1155 Oxidized Thioredoxin Reductase (TrxR) 분석 키트

BC1210/ BC1215 γ-glutamate-cysteine ligase (GCL) Assay Ki