| Product Description |
Hemoglobin (Hb or HGB) is a protein responsible for carrying oxygen in higher organisms and is the protein that makes blood appear red. Hemoglobin is composed of four chains, two alpha chains and two beta chains, each with a cyclic heme containing one iron atom. Hb is easily bound to oxygen in areas with high oxygen content; In areas with low oxygen content, it is easy to separate from oxygen. The characteristic of hemoglobin enables red blood cells to transport oxygen.
The detection principle of BIOISCO Hemoglobin Colorimetric Assay Kit is that heme in hemoglobin has a peroxidase like effect, which can catalyze the chromogenic substrate under the action of oxidants. Under acidic conditions, the product appears red with an absorption peak. The darker the red color of the product, the higher the Hb content, otherwise it is lower. The absorbance at the site is measured by an enzyme-linked immunosorbent assay (ELISA) reader, and the hemoglobin content level in the serum can be calculated through colorimetric analysis. This kit is mainly used to detect hemolytic serum samples, and can also be used to detect hemoglobin content in cell or tissue lysates or homogenates. This kit is only used in the field of scientific research and should not be used for clinical diagnosis or other purposes.
Storage conditions:
Save as required, valid for six months.
Self provided materials:
Physiological saline, 96 well plate, water bath or constant temperature box, enzyme-linked immunosorbent assay (ELISA) reader Components: | Components | H486474-100T | Storage | | Reagent (A): Hb Assay buffer | 5ml | -20℃ protect from light | | Reagent (B): chromogenic substrate | 2x100μl | -20℃ | | Reagent (C): Acid Assay buffer | 17ml | RT | | Reagent (D): Hb(1mg/ml) | 50μl | -20℃ protect from light | | Reagent (E): ddH2O | 1ml | RT |
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Matters needing attention:
1. Hb (1mg/ml) and standard working solution (13.2 μ g/ml) should be stored at -20 ℃, and repeated freezing and thawing should be avoided.
2. If there is no enzyme-linked immunosorbent assay (ELISA) reader, a regular spectrophotometer can also be used for measurement, but consideration should be given to using the minimum detection volume of the colorimetric cup as much as possible
Use a small-sized colorimetric cup.
3. After preparing one color developing working solution, it should be used up as soon as possible, so please prepare more samples for testing.
Operation steps (for reference only):
1. Preparation of testing working fluid:
① Preparation of standard working solution: Take Hb (1mg/ml) and restore it to room temperature. Accurately take it and add ddH2O to obtain the standard working solution (13.2 μ g/ml).
② Preparation of colorimetric working solution: Take Hb Assay buffer and restore it to room temperature. Dissolve 20 μ l of colorimetric substrate in 2ml Hb Assay buffer,
It is the color developing working fluid. The prepared color developing working solution should be stored at -20 ℃ and generally used up within one day.
2. Prepare samples:
① Hemolytic specimen serum: Hemolytic serum is prepared according to conventional methods and frozen at -20 ℃. Dilute with physiological saline during use.
② Cell or tissue samples: Take appropriate cell or tissue lysate, homogenize if necessary, centrifuge at low speed to obtain supernatant, and freeze at 20 ℃.
3 Hb sample addition: Set the blank hole, standard hole, and measuring hole of the 96 well plate according to the table below. The solution should be added in sequence, and attention should be paid to avoiding the generation of bubbles. If the hemoglobin content in the sample is too high, the sample dosage can be reduced or adjusted appropriately
Dilute before conducting the measurement. It is best to set parallel holes for sample testing, especially for standard samples, which should have three replicates. | Added items(μl) | Blank hole | Standard hole | Measurement hole | | ddH 2O | 8 | — | — | | Standard working fluid | — | 8 | — | | Sample to be tested | — | — | 8 | | — | — | — | — | | Color developing working fluid | 40 | 40 | 40 | | Gently mix well and incubate accurately at 37 ℃ for 10 minutes | — | — | — | | — | — | — | — | | Acid Assay buffer | 160 | 160 | 160 |
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4. Enzyme linked immunosorbent assay (ELISA) detection: The absorbance at 530nm is A530. If it cannot be detected, the absorbance can also be detected, usually within a few hours.
Calculation result:
Serum hemoglobin (mg/L)=absorbance of the test sample/absorbance of the standard sample × 13.2 × 100 (mg/L) |
