Human apo-Tf (Sigma) was treated with sodium ascorbate to remove all trace of unlabeled Fe. Apo-Tf was then loaded with 59Fe (PerkinElmer, Boston, MA, USA).
Human apo tf
Manufactured by Merck Group
Human apo-Tf is a laboratory reagent that functions as a purified form of the human protein apo-transferrin. Apo-transferrin is the iron-free form of the transferrin protein, which is responsible for the transport of iron in the human body. This product is intended for use in various research and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Ferric nitrate, by Thermo Fisher Scientific (1 mentions)
Nitrilotriacetic acid, by Merck Group (1 mentions)
Apo tf, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Feso4 7h2o, by Merck Group (1 mentions)
3 protocols using human apo tf
1
Labeled Apo-Transferrin Preparation
2
Measuring Iron Uptake by Apo-Transferrin
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The uptake of iron by Human apo-Tf was measured by the change of transferrin absorbance at 462 nm (molar extinction increase for saturated human transferrin = 4860) (Frieden and Aisen 1980 (link)). Measurements were done on a Shimadzu-UV-1601 recording spectrophotometer (Shimadzu Scientific, Columbia, MD).
Human apo-Tf (Sigma, St. Louis, MO, Product # T-1147), was dissolved in 0.05 M Tris buffer, pH 7.4, with 20 mM sodium bicarbonate. It contained < 1% iron content, as determined by iron assay with ferrozine reagent.
FPC (Rockwell, Lot #4004), and food-grade iron pyrophosphate (Dr. Paul Lohmann GmbH KG, Emmerthal, Germany) were dissolved in Tris/bicarbonate buffer. Ferric-nitrilotriacetate (Fe-NTA) was prepared from ferric nitrate (Fisher Scientific, Pittsburgh, PA) and nitrilotriacetic acid (Sigma), following the protocol of Bates et al. (Bates et al. 1967 (link)).
The apo-Tf solution (1 ml) was equilibrated at 37 °C in a quartz cuvette, the iron-complex solution (20 μL) was added, and kinetic measurements started within 10 s of mixing. Data were recorded digitally at 1–s intervals, and the reaction was monitored until steady-state measurements were achieved.
Human apo-Tf (Sigma, St. Louis, MO, Product # T-1147), was dissolved in 0.05 M Tris buffer, pH 7.4, with 20 mM sodium bicarbonate. It contained < 1% iron content, as determined by iron assay with ferrozine reagent.
FPC (Rockwell, Lot #4004), and food-grade iron pyrophosphate (Dr. Paul Lohmann GmbH KG, Emmerthal, Germany) were dissolved in Tris/bicarbonate buffer. Ferric-nitrilotriacetate (Fe-NTA) was prepared from ferric nitrate (Fisher Scientific, Pittsburgh, PA) and nitrilotriacetic acid (Sigma), following the protocol of Bates et al. (Bates et al. 1967 (link)).
The apo-Tf solution (1 ml) was equilibrated at 37 °C in a quartz cuvette, the iron-complex solution (20 μL) was added, and kinetic measurements started within 10 s of mixing. Data were recorded digitally at 1–s intervals, and the reaction was monitored until steady-state measurements were achieved.
3
Retinal Explant Culture for Iron Exposure and Transferrin Therapy
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Adult Wistar rat retinas were isolated from fresh enucleated eyes, divided into four parts, and transferred to 0.2-mm polycarbonate membranes (Millipore) with the GCL facing the membrane (Fig. 2 ). Mouse retinal explants were isolated as indicated above, with one explant corresponding to one eye. The insert was placed into a six-well culture plate containing Dulbecco’s modified Eagle’s medium with l -glutamine and 5% serum (Thermo Fisher Scientific) corresponding to day 0. To analyze the effects of iron exposure on retinal explants, 30 μl of medium containing FeSO4 (7H2O; Sigma-Aldrich) was added to the PR side of the explants (upper chamber). To explore TF’s therapeutic effects, the medium in the upper chamber was removed after 2 days. PBS was added for washing, and 30 μl of medium containing human apoTF (0.5, 5, or 50 mg/ml; Sigma-Aldrich) was placed on the retinas. LDH release and enzyme-linked immunosorbent assay (ELISA) directed against TF were measured in medium collected from the lower chamber as described previously (12 (link), 13 (link)). Histology, iron staining, immunohistochemistry, and Western blotting were performed on explants as described below and in the time workflow legends.
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