Total proteins were extracted by using lysis buffer (Beyotime), separated by SDS/PAGE gels and transferred to the membranes (Millipore, MA, USA). After hybridization, the membranes were incubated with the primary antibody at 4 °C condition for one night. In this study, the primary antibodies used were: β‐actin (IgG, CST, USA), ferritin (IgG, Abcam, Cambridge, Britain), ferritin (IgG, Abcam), DMT1 (IgG, Abcam), Fpn1 (IgG, Abcam) and TfR1 (IgG, Abcam). The next day, the membranes were immunoblotted with a secondary antibody (CST, Danvers, MA, USA) at room temperature for 60 min. The intensity of protein bands was analyzed using imagej software (NIH, USA).
Ferritin
Manufactured by Abcam
Sourced in United States, United Kingdom
Ferritin is a protein that stores iron in cells. It is found in most tissues in the body, with the highest concentrations in the liver, spleen, and bone marrow. Ferritin plays a crucial role in the regulation and distribution of iron, which is essential for various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Abcam (3 mentions)
C reactive protein (crp), by R&D Systems (3 mentions)
Transferrin receptor, by Abcam (2 mentions)
Erythropoietin, by R&D Systems (2 mentions)
Insulin, by Mercodia (2 mentions)
Adiponectin, by R&D Systems (2 mentions)
Gdf 15, by R&D Systems (2 mentions)
Interleukin 6 (il 6), by R&D Systems (2 mentions)
Acetaminophen, by Sekisui (2 mentions)
Hepcidin, by Abcam (1 mentions)
Interleukin il 1β, by R&D Systems (1 mentions)
Interleukin 6 (il 6), by BioLegend (1 mentions)
Tnf α, by BD (1 mentions)
Transferrin, by Abcam (1 mentions)
Quantichrom iron assay kit, by BioAssay Systems (1 mentions)
Hemoglobin colorimetric assay kit, by Cayman Chemical (1 mentions)
Mem pertm plus membrane protein extraction kit, by Thermo Fisher Scientific (1 mentions)
Phospho pi3k, by Abcam (1 mentions)
T pi3k, by Abcam (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
27 protocols using ferritin
1
Western Blot Analysis of Iron-Related Proteins
2
Immunohistochemistry Analysis of Iron Regulatory Proteins
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The paraffin sections were deparaffinized in xylene, rehydrated in distilled water, treated with 3% H2O2 for 30 min at 37°C to block endogenous peroxidase activity and washed with 0.1M PBS 3 times. Nonspecific binding was blocked by 10% goat serum for 40 min at 37°C and was followed by incubation overnight at 4°C with antibodies against the following: DMT1 with IRE (#NRAMP21-A; 1:2,000) and DMT1 without IRE (#NRAMP23-A; 1:2,000) from Alpha Diagnostics International; Transferrin antibody (N3C3; 1:250) from GeneTex;TfR1 (1:100) from Thermo; FPN1 (1: 2,000; #MTP11-A) from Alpha Diagnostics International, Ceruloplasmin (1:50) from Origene; Hephaestin (1:200) from Alpha Diagnostics International; Ferritin(1:200) from Abcam; Hepcidin (1:50) from Abcam. On the following day, sections were washed in 0.1M PBS and incubated with biotinylated goat anti-rabbit IgG for 25 min. Finally, the sections were washed in 0.1M PBS, stained with DAB, dehydrated using graded ethanol, and cleared using xylene. Images of positive staining in the frontal cortex region were captured at 20× magnification by a Nikon 80i microscope and analyzed using ipwin32 software to measure Integral optical density (IOD).
3
Serum Biomarkers of Iron Metabolism
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Levels of hepcidin, ERFE (MyBioSource, San Diego, CA, USA), ferritin (Abcam), interleukin- (IL-) 1β (R&D), IL-6 (BioLegend, San Diego, CA, USA), and tumor necrosis factor-α (TNF-α) (BD Biosciences, Franklin Lakes, NJ, USA) in the serum samples were determined using ELISA commercial kits according to the manufacturer's instructions.
4
Mouse Serum Ferritin ELISA
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ELISA for node mouse serum Ferritin (ab157713, Abcam) was performed according to the manufacturer’s instructions.
5
Plasma Biomarkers Quantification
6
Quantifying Tissue Iron and Plasma Markers
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Tissue non-heme iron concentration in renal cortex, outer medulla and liver was measured by a microplate colorimetric assay following acid extraction and reaction with bathophenanthroline reagent as described by Grundy et al.42 (link) Ferric citrate (AvantorTM Performance Materials, Center Valley, PA) was used to make iron standards for the assay. Iron concentrations were normalized to wet tissue weight. Plasma total iron was measured using the Quantichrom™ Iron Assay Kit (BioAssay Systems, Hayward, CA). BUN and plasma creatinine were measured using QuantichromTM urea and creatinine assays, respectively (BioAssay Systems). Commercially available ELISAs were used to measure urine concentrations of transferrin (ADI, San Antonio, TX), ferritin (abcam, Cambridge, UK; also used for plasma) and albumin (Exocell, Philadelphia, PA). Plasma autoantibodies were measured using a mouse anti-dsDNA IgG-specific ELISA kit (ADI). Hemoglobin concentrations were measured in lysed red blood cells and in plasma using Cayman Chemical’s Hemoglobin Colorimetric Assay Kit (Ann Arbor, MI).
7
Protein Extraction and Western Blot Analysis
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Cells were lysed by RIPA. After being shocked and ice-bath for 30 min, cells were centrifuged for 20 min at 22,000 rpm at 4°C. The supernatants were collected. Cell membrane protein and cytoplasmic protein were separated using Mem-PERTM Plus Membrane Protein Extraction Kit (Thermo). The concentration of protein was accessed using BCA. 20–40 μg protein was separated by 8–12.5% SDS-PAGE, then transferred to polyvinylidene fluoride membrane (Immobilon-P) using Trans-Blot Turbo Transfer System (Bio-Rad). DMT1, IRP1, IRP2, TfR1, TfR2, ferritin, T-PI3K, phospho-PI3K antibodies were obtained from Abcam. T-STAT3 (0.1 mg/ml), phospho-STAT3 tyr705 (1 mg/ml) were acquired from R&D. T-ERK, phospho-ERK, pan-AKT, phospho-AKT (1:1000) were obtained from Cell signaling technology. GAPDH and β-actin antibodies were purchased from Biotime. After being cultured with primary antibodies overnight at 4 °C, proteins were cultured with the HRP-labeled secondary antibody (KPL). Proteins were visualized using enhanced chemiluminescence (Pierce). All experiments were performed in triplicate.
8
Western Blot Analysis of Iron Metabolism Proteins
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Total proteins were extracted by the total protein extraction kit (Keygene Biotech). Western blot assays were performed and analyzed as before.[22 ] Antibodies used in this study were β‐actin (Cat. ab8227; Abcam), SLC46A1 (Cat. ab25134; Abcam), STEAP3 (Cat. ab180770; Abcam), DMT1 (Cat. ab55735; Abcam), TfR1 (Cat. ab84036; Abcam), FPN (Cat. ab85370; Abcam), HAMP (Cat. ab30760; Abcam), and ferritin (Cat. ab75973; Abcam). The IRDye secondary antibody (LI‐COR) was used, and immunoblots were scanned by Odyssey dual‐color infrared fluorescence imaging system. The grayscale of each band was obtained from Odyssey software.
9
Western Blotting Analysis of Cellular Proteins
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Total proteins (20 μg) were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane for Western blotting. The membranes were blocked with 5% skim milk (Sigma) in Tris–buffered saline for 1 h at room temperature and then incubated with various primary antibodies overnight at 4°C: tumor necrosis factor (TNF)-α, cluster of differentiation 11b (CD11b), heme oxygenase (HO)-1, ferritin, and tubulin (all 1:1,000; Abcam, Cambridge, MA, United States); NAD(P)H quinone dehydrogenase 1 (NQO1), B-cell lymphoma-2 (Bcl-2)-associated X protein (BAX), transferrin, and actin (all 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, United States); p62 and microtubule-associated protein 1A/1B light chain (LC) 3B (all 1:1,000; Cell Signaling Technology, Danvers, MA, United States); and glial fibrillary acidic protein (GFAP) (1:5,000; Agilent Technologies, Santa Clara, CA, United States). Further, the blots were probed with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Santa Cruz Biotechnology) and visualized using SuperSignal West Femto Substrate Maximum Sensitivity Substrate (Thermo Fisher Scientific). A ChemiDoc image analyzer was used to detect immunoblotted bands (Bio-Rad, Hercules, CA, United States).
10
Western Blot Analysis of Mitochondrial Proteins
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Proteins were isolated with RIPA buffer plus protease inhibitor cocktail (Sigma, P8340) and a tissue grinder for 5 s at 25,000 rpm (IKA; T25 digital Ultra Turrax, Staufen, Germany). Protein concentration was determined with the Pierce BCA assay kit (ThermoFisher 23225, Waltham, MA, USA). Proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; ThermoFisher), transferred to PVDF membranes (Millipore, Burlington, MA, USA) and blotted with the following primary antibodies. HO-1 (in-house, need ref); TOM20 (Cell Signaling 42406, Danvers, MA, USA); total rodent oxphos antibody cocktail (Abcam ab110413, Cambridge, UK); calnexin (Enzo, ADI-SPA-860-F, Farmingdale, NY, USA); transferrin receptor (ThermoFisher 13-6800); ferritin (Abcam ab75973). Horseradish peroxidase conjugated secondary antibodies were used for visualization with Luminata Crescendo (EMD Millipore) or SuperSignal West Femto substrate (ThermoFisher) using the ChemiDoc Touch Imaging System (BioRAD, Hercules, CA, USA).
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