Solarbio Cell Iron Content Assay Kit 50T | 48S

Cell Iron Content Assay Kit

注記: 予測する必要がある 2-3 正式な決定前の大きな差異のサンプル. 操作装置: 分光光度計

カタログ番号: BC5310

サイズ:50T/48S

コンポーネント:

抽出液: 液体 30 mL×1. Store at 2-8℃.

試薬I: 液体 15 mL×1. Store at 2-8℃. 試薬II: 液体 35 mL×1. Store at 2-8℃. 試薬Ⅲ: 液体 6 mL×1. Store at 2-8℃.

標準: 液体 1 mL×1. Store at 2-8℃. 1 μmol/mL of Fe3+ standard solution. Add 200μL distilled water to 200μL Fe3+ standard solution of 1 使用前のμmol/ml, and mix well to form the Fe3+ standard solution of 0.5 μmol/mL.

製品説明:

Iron is one of the essential trace elements in the human body, which is the main component of hemoglobin, myoglobin, cytochrome, and other enzyme systems. Iron can assist in the transport of oxygen and promote fat oxidation. Iron deficiency can easily cause anemia, and metabolic disorders, and affect the immune function of the body.

Fe3+ is reduced by hydroxylamine hydrochloride to Fe2+. Fe2+ could react with Phenanthroline Monohydrate to form a kind of orange compound under weak acid conditions, which has an absorption peak at 510 nm. According to the measure, absorbance at 520 nm can reflect cell iron concentration.

必要だが提供されていない試薬と装置:

分光光度計, 卓上遠心分離機, 調整可能なピペット, 1mLガラスキュベット, cell ultrasonic crusher, 氷, 蒸留水.

手順:

1. サンプルの準備:
2. 細菌や細胞を遠心管に集めます, and discard the supernatant after centrifugation. According to the proportion of bacteria or cell number (104): 抽出液量 (mL) の 500-1000-1 抽出する. それは提案されています 5 million bacteria or cells amount to 0.5mL of Extract solution. Split the bacteria or cell with ultrasonication (氷の上に置かれた, 超音波出力200W, 作業時間3秒, インターバル7秒, 繰り返す 30 回). で遠心分離します。 8000 グラムのための 10 4℃で3分間不溶物を除去, and take the supernatant on ice before
3. Ultrasonication working time could be prolonged properly if samples are fungi, 細菌, or other microorganisms with cell

Ⅱ. 決定 手順:

1. 分光光度計を予熱します。 30 分, 波長を調整して 510 nm, and set zero with distilled
2. Add reagents with the following:

試薬 (μL)	試験管 (で)	ブランクチューブ (AB)	標準チューブ (として)
サンプル	100	–	–
蒸留水	–	100	–
標準溶液 (0.5 μmol/mL)	–	–	100
試薬I	200	200	200
試薬Ⅱ	600	600	600
試薬Ⅲ	100	100	100
十分に混ぜ合わせてください, and place at 25℃ for 10 分. Take 1mL of reaction solution in 1mL glass cuvette. 吸光度を測定します。 510 nm, recorded as AT, AB, and AS. ΔAT = AT-AB, ΔAS=AS-AB. Blank tubes and standard tubes only need to be tested once or twice.

Ⅲ. Cell Iron Content 計算

• Bacteria/cells number

Cell iron content (ng/104 cell) =(Cs×ΔAT÷ΔAS)×VE×103×55.845÷500=27.922×ΔAT÷ΔAS

2) タンパク質 集中

Cell iron content (ng/mg prot)

=(Cs×ΔAT÷ΔAS)×VE×103×55.845÷(Cpr×VE)=27922.5×ΔAT÷ΔAS÷Cpr

Cs: Concentration of Fe3+ standard solution, 0.5μmol/mL;

ve: 抽出液量, 0.5 mL;

103: Unit conversion factor, 1 μmol=103 nmol;

55.845: Relative molecular mass of Fe, 55.845ng/nmol; 500: 細菌または細胞の総数, 5 百万;

心肺蘇生法: 上清サンプルのタンパク質濃度, mg/mL.

注記:

1. If ΔAT＜01, it is recommended to increase the sample supernatant size before determination. If AT＞1, it is recommended to dilute the sample supernatant with distilled water before determination. And modify the calculation formula.
2. Sample supernatant protein concentration needs to be measured by yourself if cell iron content is calculated by protein

実験例:

1. 取る 5 百万個の細胞, 追加 0.5 抽出液 mL, and split with ultrasonication. Centrifuge and take the supernatant. Then operate according to the determination steps, calculate ΔAT=AT-AB= 0.171- 0.000=0.171, ΔAS=AS-AB=0.573-0.000=0.573. The result is calculated according to cell numbers: Cell iron content (ng/104 cells) =27.922×ΔAT÷ΔAS=8.333ng/104 cell

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