Heme Assay Kit

Catalog No. ABIN1000266

Product Details

Target: Heme

Application: Biochemical Assay (BCA)

Sample Type: Blood, Serum, Plasma, Urine

Specificity: 0.6 μM

Characteristics: Sensitive and accurate. Linear detection range 0.6 - 125 µM heme in 96-well plate assay.
Simple and high-throughput. The mix-and-read procedure involves addition of a single working reagent and reading the optical density. Can be readily automated as a high-throughput assay in 96-well plates for thousands of samples per day.
Safety. Reagents are non-toxic.
Versatility. Assays can be executed in 96-well plate or cuvet.

Components: Reagent: 50 mL. Calibrator: 10 mL (equivalent to 62.5 µM heme).

Material not included: Pipeting devices and accessories. Clear-bottom 96-well plates (e.g. Corning Costar) and plate reader. Cuvets and spectrophotometer.

Application Details

Application Notes: Direct Assays: total heme in blood, serum, plasma, urine, heme- carrying enzymes.
Pharmacology: effects of drugs on heme metabolism.
Drug Discovery: HTS for drugs that modulate heme levels.

Protocol: Procedure using 96-well plate:
1. Blank and Calibrator. Pipette 50 µL water (Blank) and 50 µL Calibrator into wells of a clear bottom 96-well plate. Transfer 200 µL water into the blank and Calibrator wells. The diluted calibrator is equivalent to 62.5 µMheme.
2. Samples. Serum and plasma samples can be assayed directly (n = 1). Blood samples should be diluted 100-fold in distilled water (n = 100). Transfer 50 µL samples into wells (important: avoid bubble formation during the pipetting steps). Add 200 µL Reagent to sample wells and tap plate lightly to mix.
3. Incubate 5 min at room temperature. Read OD at 380-420nm (peak 400nm).

Procedure using cuvette:
1. Transfer 100 µL sample and 1000 µL Reagent into a cuvet and tap lightly to mix. Read OD at 380-420nm (peak 400 nm) against water.
2. Transfer 100 µL Calibrator and 1000µL water to cuvet. Read OD at 400nm against water.

Calculation of Results:

Subtract blank OD (water) from the Calibrator and Sample OD values.
Conversions: 1mg/dL heme equals 15.3 µM, 0.001% or 10 ppm.

Restrictions: For Research Use only

Handling

Storage: 4 °C

References

Ndisang, Lane, Syed, Jadhav: "Up-regulating the heme oxygenase system with hemin improves insulin sensitivity and glucose metabolism in adult spontaneously hypertensive rats." in: Endocrinology, Vol. 151, Issue 2, pp. 549-60, (2010) (PubMed).

Aldag, Gromov, García-Rubio, von Koenig, Schlichting, Jaun, Hilvert: "Probing the role of the proximal heme ligand in cytochrome P450cam by recombinant incorporation of selenocysteine." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 106, Issue 14, pp. 5481-6, (2009) (PubMed).

Huang, Becker, Whitnall, Suryo Rahmanto, Ponka, Richardson: "Elucidation of the mechanism of mitochondrial iron loading in Friedreich's ataxia by analysis of a mouse mutant." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 106, Issue 38, pp. 16381-6, (2009) (PubMed).

Ndisang, Jadhav: "The heme oxygenase system attenuates pancreatic lesions and improves insulin sensitivity and glucose metabolism in deoxycorticosterone acetate hypertension." in: American journal of physiology. Regulatory, integrative and comparative physiology, Vol. 298, Issue 1, pp. R211-23, (2009) (PubMed).

Zwart, Kala, Smith: "Body iron stores and oxidative damage in humans increased during and after a 10- to 12-day undersea dive." in: The Journal of nutrition, Vol. 139, Issue 1, pp. 90-5, (2008) (PubMed).

Pamplona, Ferreira, Balla, Jeney, Balla, Epiphanio, Chora, Rodrigues, Gregoire, Cunha-Rodrigues, Portugal, Soares, Mota: "Heme oxygenase-1 and carbon monoxide suppress the pathogenesis of experimental cerebral malaria." in: Nature medicine, Vol. 13, Issue 6, pp. 703-10, (2007) (PubMed).

Target Details

Target: Heme

Background: Quantitative determination of heme by colorimetric (400nm) method.
Procedure: 5 min.

Heme is one important member of the porphyrin family. It is synthesized in both mitochondria and cytoplasm, and is a key prosthetic group for various essential proteins such as hemoglobin, cytochromes, catalases and peroxidases. Heme determination is widely practiced by researchers of various blood diseases. Simple, direct and automation-ready procedures for measuring heme concentration are becoming popular in Research and Drug Discovery. This Heme Assay Kit is based on an improved aqueous alkaline solution method, in which the heme is converted into a uniform colored form. The intensity of color, measured at 400 nm, is directly proportional to the heme concentration in the sample. The optimized formulation substantially reduces interference by substances in the raw samples and exhibits high sensitivity.