ソーラーバイオ酸化グルタチオン (GSSG) コンテンツアッセイキット

酸化グルタチオン (GSSG) コンテンツアッセイキット

注記: テスト前に予測のために 2 つまたは 3 つの異なるサンプルを取得します.

操作装置: 分光光度計/マイクロプレートリーダー

カタログ番号: BC1185

サイズ:100T/96S

コンポーネント:

試薬I:100 mL×1. 4℃で保存. 試薬II: 130 μL×1. 4℃で保存. 試薬Ⅲ: 20 mL×1. 4℃で保存. 試薬IV:2.5 mL×1. 4℃で保存.

試薬V: Powder ×1. 4℃で保存. で溶かす 2.5 溶液を使用する場合は蒸留水 mL, then split into smaller packages, -20℃で保存.

試薬VI:12.5 μL×1. 4℃で保存. Prepare Reagent VI and distilled water according to the sample size at the ratio of 1:20 (V: V) 使用前に.

標準: 粉 10 mg×1. 4℃で保存.

製品説明

酸化グルタチオン(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.

技術仕様

Minimum Detection Limit：3.211 μg/mL

線形範囲：3.9-125 μg/mL

必要だが提供されていない試薬と装置.

Analytical balance, モルタル/ホモジナイザー, refrigerated centrifuge, 水浴, 調整可能なピペット, spectrophotometer/ microplate reader, マイクロガラスキュベット/96ウェル平底プレート.

手順

私. サンプル preparation

1. 1. 組織サンプル

Wash fresh tissues with PBS for twice, then add 0.1 g of sample into the homogenizer (the homogenizer has been rinsed with Reagent I and placed on ice before use). 追加 1 試薬I mL (the proportion of tissue and reagents can be kept constant), 氷上で完全に粉砕する (using liquid nitrogen will have a better grinding effect). で遠心分離します。 8000 ×g and 4℃ for 10 分, take the supernatant and place it at 4℃ for test. (The supernatant can be stored at -80℃ for 10 days.)

2. Blood sample

プラズマ: Sample is centrifuged at 600 ×g and 4℃ for 10 分. Absorbing the upper plasma into another tube add with same volume Reagent I. で遠心分離します。 8000 ×g and 4℃ for 10 分, 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 分. Discarding the upper plasma, wash with treble volume of PBS for 3 回 (mix blood cell with PBS centrifuge at 600 ×gの場合 10 分), add equal volume of Reagent I. After mixing, it is placed at 4℃ for 10 分. で遠心分離します。 8000 ×gの場合 10 分, take the supernatant and place it at 4℃ for test. (The supernatant can be stored at -80℃ for 10 days.)

3. Cell sample

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

Ⅱ. 手順

1. 予熱分光光度計/マイクロプレートリーダー 30 分, 波長を調整して 412 nm, set the counter to zero with distilled
2. Preheat Reagent II in water bath for 30 分: 37℃ (mammal cell) または25℃ (他の種).
3. The standard dilution: dissolve standard with 1 蒸留水 mL (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. 追加 20 μL of diluted standard or sample to 0.5 mL centrifuge tube, 追加 1 試薬 II μL, incubate at 37 ℃ for 30 minutes after mixing.
5. Make standard curves

After the incubation, 追加 140 μL of Reagent III, 20 試薬 IV μL, 20μL of Reagent V, そして 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 (バツ) and concentration is the ordinate (y), making the standard curve.

追加 140 μL of Reagent III, 20 試薬 IV μL, 20 μL of Reagent V, そして 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. 計算

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

(μg/ml).

• タンパク質濃度

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

• サンプル重量

GSSH (μg /g)= y×Vrv÷(Vrv÷Vsv×W)= y÷W

• セラマウント

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

• Solution volume

GSSH (μg / mL)= y×Vrv/Vs= 2y

N: 細胞量,106；

ロープ: 総反応量, 0.203 mL；

VSv: The volume of supernatant was added into the reaction system, 20 μL=0.02 mL； W: サンプル重量, g;

心肺蘇生法: 上清タンパク質濃度, mg/mL.

ノート:

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 そして 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.

Reference:

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

関連製品：

BC1170/ BC1175 Reduced Glutathione（GSH）Assay Kit

BC1190/ BC1195 Glutathione Peroxidase Assay Kit

BC0350/ BC0355 Glutathione S-transferase(GST) 活性アッセイキット

BC1160/ BC1165 Glutathione Reductase (GR) アッセイキット