Recombinant Human Hemopexin Protein, CF Summary
Thr24-His462, with a C-terminal 6-His tag
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CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|Formulation||Supplied as a 0.2 μm filtered solution in MES and NaCl.|
|Shipping||The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Assay Buffer A: 0.1 M Sodium Borate, pH 9.0
- Assay Buffer B: 5 mM NaOH in 50% (v/v) Glycerol and 50% (v/v) Methanol solution
- Assay Buffer C: 50% Assay Buffer A, 50% Assay Buffer B
- Recombinant Human Hemopexin (rhHemopexin) (Catalog # 4490-HP)
- Protoporphyrin IX (PPP-IX) (Sigma, Catalog # P8293) Prepare 10 mM Stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhHemopexin to 60 µg/mL in Assay Buffer A.
- Dilute PPP-IX to 640 µM in Assay Buffer B.
- Dilute the 640 µM PPP-IX to 320 μM in Assay Buffer A.
- Prepare a curve of PPP-IX in Assay Buffer C. Make the following serial dilutions starting with the dilution made in the previous step: 320, 160, 80, 40, 20, 10, 5, 2.5, and 1.25 µM.
- Mix equal volumes of the PPP-IX curve dilutions and the 60 µg/mL rhHemopexin. Include a control containing Assay Buffer C and the 60 µg/mL rhHemopexin.
- Incubate mixtures at room temperature for 30 minutes.
- Load 100 µL of reaction mixtures and control into a plate.
- Read at excitation and emission wavelengths of 280 nm and 340 nm (top read), respectively in endpoint mode.
- Plot the curve to obtain the concentration of PPP-IX that results in 50% decrease in the fluorescence signal from the control.
- rhHemopexin: 30 µg/mL (0.6 µM)
- PPP-IX curve: 160, 80, 40, 20, 10, 5, 2.5, 1.25, and 0.625 µM
Hemopexin (HPX) is a 60 kDa plasma glycoprotein with two four-bladed beta -propeller folds. This structural motif has been found in other proteins including collagenases and provides sites for protein-protein interactions (1-3). The liver is the major synthesizing organ. Expression in the central nervous system, in the retina, and in peripheral nerves has also been observed. Hemopexin belongs to the family of the acute-phase proteins whose synthesis is induced after an inflammatory event. Hemopexin participates in maintaining and recycling the iron pool by utilizing its high binding affinity toward heme composed of protoporphyrin IX and iron. It also functions in preventing oxidation caused by heme after hemolysis. Hydrophobic heme molecules can intercalate into lipid membranes and participate in the oxidation of lipid membrane components through the Fenton reaction resulting in lipid peroxidation. Hemopexin undergoes a conformational change upon the binding of heme. The conformational change allows hemopexin to interact with a specific receptor, forming a complex which is then internalized. In the plasma, it is likely that heme binds albumin (35-55 g/L) first because of the higher concentration of albumin in plasma than hemopexin (0.5-1.2 g/L), and is then transferred to hemopexin, which has a much higher affinity (Kd ~1 pM) toward heme. Heme concentrations in plasma increase after hemolysis, which is associated with several pathological conditions such as reperfusion injury and ischemia.
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