Solarbio Cell Iron Content Assay Kit 50T | 48S

Cell Iron Content Assay Kit

Notiz: Es ist notwendig, vorherzusagen 2-3 große Differenzproben vor der formalen Bestimmung. Betriebsausrüstung: Spektrophotometer

Katze nein: BC5310

Größe:50T/48S

Komponenten:

Lösung extrahieren: Flüssig 30 ml×1. Store at 2-8℃.

Reagenz I: Flüssig 15 ml×1. Store at 2-8℃. Reagenz II: Flüssig 35 ml×1. Store at 2-8℃. Reagenz III: Flüssig 6 ml×1. Store at 2-8℃.

Standard: Flüssig 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 μmol/mL before use, and mix well to form the Fe3+ standard solution of 0.5 μmol/ml.

Produktbeschreibung:

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.

Erforderliche, aber nicht bereitgestellte Reagenzien und Ausrüstung:

Spektrophotometer, Tischzentrifuge, verstellbare Pipette, 1ML Glass Cuvette, cell ultrasonic crusher, Eis, destilliertes Wasser.

Verfahren:

1. Probenvorbereitung:
2. Sammeln Sie Bakterien oder Zellen in die Zentrifugenröhre, and discard the supernatant after centrifugation. According to the proportion of bacteria or cell number (104): Lösungsvolumen extrahieren (ml) von 500-1000-1 extrahieren. Es wird vorgeschlagen, dass 5 million bacteria or cells amount to 0.5mL of Extract solution. Split the bacteria or cell with ultrasonication (auf Eis gelegt, Ultraschallleistung 200W, Arbeitszeit 3s, interval 7s, wiederholen 30 mal). Zentrifuge bei 8000 G für 10 Minuten bei 4 ℃, um unlösliche Materialien zu entfernen, and take the supernatant on ice before
3. Ultrasonication working time could be prolonged properly if samples are fungi, Bakterien, or other microorganisms with cell

II. Bestimmung Verfahren:

1. Das Spektrophotometer vorheizen 30 Protokoll, Passen Sie die Wellenlänge an 510 nm, and set zero with distilled
2. Add reagents with the following:

Reagens (μL)	Reagenzglas (BEI)	Leeres Rohr (AB)	Standardrohr (ALS)
Probe	100	–	–
Destilliertes Wasser	–	100	–
Standardlösung (0.5 μmol/ml)	–	–	100
Reagenz I	200	200	200
Reagenz ⅱ	600	600	600
Reagenz ⅲ	100	100	100
Gründlich mischen, and place at 25℃ for 10 Protokoll. Take 1mL of reaction solution in 1mL glass cuvette. Messen Sie die Absorption bei 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 Berechnungen

• Bacteria/cells number

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

2) Protein Konzentration

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: Lösungsvolumen extrahieren, 0.5 ml;

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

55.845: Relative molecular mass of Fe, 55.845ng/nmol; 500: Gesamtzahl von Bakterien oder Zellen, 5 Million;

Cpr: Supernatant sample protein concentration, mg/ml.

Notiz:

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

Experimentelles Beispiel:

1. Nehmen 5 Millionen Zellen, hinzufügen 0.5 ml Extraktlösung, 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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