1-DE was performed by resolving the protein on a uniform 12.5% gel and then electrotransferring to nitrocellulose membranes at 150 mA for 2 h. The membranes were subsequently blocked with 5% (w/v) non-fat milk for 1 h at 25 °C. The membranes were probed with anti-FLAG (F1804; Sigma), anti-actin (A0480; Sigma) and anti-ferritin (ab7332; abcam), anti-CaFer1 (customized against 54–66 (GVLFEPFEEVKKE) amino acid sequence, anti-FBA (AS08294; Agrisera) and anti-thioredoxin (T0803; Sigma-Aldrich) antibodies diluted to 1:1000 in Tris-buffered saline (TBS). The antibody bound proteins were detected by incubation with horseradish peroxidase-conjugated anti-rabbit IgG, and the bands were visualized using the ECL detection system (GE Healthcare).
Anti ferritin
Manufactured by Abcam
Sourced in United States, China
Anti-ferritin is a laboratory reagent used to detect and measure ferritin, an iron-storing protein, in biological samples. It provides a quantitative assessment of ferritin levels, which can be useful in the diagnosis and monitoring of conditions related to iron metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Anti gm130, by BD (3 mentions)
Anti actin, by Merck Group (2 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (2 mentions)
Fluorescent dye conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti iba1, by Fujifilm (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Anti cd81, by Thermo Fisher Scientific (2 mentions)
Anti flotillin 1, by BD (2 mentions)
Anti tsg101, by GeneTex (2 mentions)
Anti hsp90, by Enzo Life Sciences (2 mentions)
Tris gels, by Bio-Rad (2 mentions)
Anti lactoferrin, by Santa Cruz Biotechnology (2 mentions)
Anti mhc 1, by LifeSpan BioSciences (2 mentions)
Anti calnexin, by Santa Cruz Biotechnology (2 mentions)
Anti transferrin, by Santa Cruz Biotechnology (2 mentions)
Anti tfr, by Abcam (2 mentions)
Peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Primary antibody dilution buffer, by Beyotime (2 mentions)
Nmdar2a, by Abcam (2 mentions)
16 protocols using anti ferritin
1
Western Blot Analysis of Cellular Proteins
2
Quantifying Ferritinophagy in GFP-LC3 Mice
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For ferritinophagy analysis, BMCs of GFP-LC3 mice were stained with HSC markers and followed by flow cytometric detection of GFP-LC3. For ferritin detection, cells were permeabilized with Permeabilization buffer (eBioscience) in the presence of anti-Ferritin (Abcam) antibody at room temperature for 45 min and stained with fluorescent dye conjugated secondary antibodies (Thermo Fisher Scientific) at room temperature for another 30 min and finally analyzed by flow cytometry.
3
Western Blotting Protocol for mBMSCs
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Protein was extracted from mBMSCs using RIPA lysis buffer (Biosharp, Beijing, China) and determined using Pierce BCA Protein Assay kit (Thermo Fisher Scientific), according to the instructions. The proteins were separated using a 10% SDS-PAGE gel and subsequently transferred to the polyvinylidene fluoride membrane. After sealing polyvinylidene fluoride membrane in 5% defatted milk, primary antibody was added and incubated at 4 °C overnight. All the primary antibodies we used in this study were as follows: anti-Ferritin (1:1000; Abcam) and anti-β-actin (1:2000; Abcam). Finally, the membranes were incubated with anti-rabbit secondary antibody (1:5000; Abcam) for 1 h at room temperature after washing with PBST. The bands were scanned using Tanon 5200 Automatic chemiluminescence image analysis system (Shanghai, China).
4
Antibody Immunodetection Assay Protocol
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The following antibodies were used for the immunoblot and immunofluorescence assays: anti-GFP (Santa Cruz Biotechnology, #sc-9996), anti-Wipi3 (Thermo Fisher Scientific, #PA5-50864), anti-Atg5 (Sigma, #A0731), anti-Lamp2 (Abcam, #ab13524), anti-RFP (MBL, #M204-3), anti-Flag (MBL, #PM020), anti-HA (Santa Cruz Biotechnology, #sc-7392), anti-Lamp1 (abcam, #ab24170), anti-VSVG (KeraFAST, #EB0010), anti-GS28 (BD, #611184), anti-GM130 (BD, #610823), anti-Tom20 (Santa Cruz Biotechnology, #sc-11415), anti-Myc (Santa Cruz Biotechnology, #sc-40), anti-Rab9 (Sigma, #R5404), anti-His (Nacalai Tesque, #04428-26), anti-LC3 (nanoTools, #0231-100), anti-p62 (MBL, #PM045), anti-Wipi2 (abcam, #ab105459), anti-actin (Millipore, #MAB1501), anti-GST (Santa Cruz Biotechnology, #sc-138), anti-Atg7 (CST, #2631), anti-GAPDH (EMD Millipore, #MAB374), anti-calbindin D-28k (Swant, #300), anti-calbindin D-28k (Sigma, #C2724), anti-GFAP (Cell Signaling Technology #12389), anti-Iba1 (Wako, #019-19741), anti-ubiquitin (CST, #3933), anti-ceruloplasmin (BD, #611488), anti-ferritin (abcam, #ab69090), anti-ferroportin (Novus, #NBP1-21502) and anti-DMT1 (Proteintech, #20507-1-AP). Enzymes used for recombinant DNA techniques were purchased from Takara Bio, TOYOBO, and New England BioLabs. All other reagents were obtained from Nacalai Tesque.
5
Antibody Acquisition for TLR4, BAX, HO1
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The antibodies used in this study were obtained as follows: rabbit anti-toll-like receptor 4 (TLR4), anti-BAX, and goat anti-Actin were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); rabbit anti-heme oxygenase-1 (HO1) and anti-ferritin were purchased from Abcam (Abcam, MA, USA); and all secondary antibodies were purchased from Santa Cruz Biotechnology.
6
Protein Separation and Detection via SDS-PAGE and Western Blotting
7
Immunoblotting Analysis of Mitochondrial Proteins
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Proteins from lysates were prepared and resolved by 12% SDS-PAGE at 100 V, transferred for 1.5 h at 250 mA onto Nitrocellulose membranes, and analysed by immunoblotting as described previously56 (link). The information for primary antibodies is as follows: anti-ferritin (cat# 69090), SdhA (cat# 137040), and SdhB (cat# 178423) from Abcam (Cambridge, MA, USA), anti-Xod (cat# 55156-1-AP), citrate synthase (cat# 16131-1-AP), aconitase 2 (Aco2) (cat# 11134-1-AP), Ndufs1 (cat# 12444-1-AP), Ndufs3 (cat# 15066-1-AP), Uqcrc1 (cat# 21705-1-AP), and Uqcrfs1 (cat# 1843-1-AP) from Proteintech Group Inc. (Chicago, IN, USA), anti-Actin (cat# BM0627) from Boster (Wuhan, China), anti-Tubulin (cat# T0198) from Sigma-Aldrich (St. Louis, MO, USA), anti-TfR1 antibody (cat# 136800) from Zymed (San Francisco, CA, USA), anti-IscU, Fech, Irp1, and Irp2, anti-Fxn (self-made)31 (link), anti-CytC (cat# 1896-1) from Epitmics (Burlingame, CA, USA), anti-Vdac (cat# 4661S) from Cell Signaling Technology Inc (Shanghai, China).
8
Immunoblot Analysis of Iron Regulatory Proteins
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Tissue was homogenized in lysis buffer containing 1% Triton X-100, 20 mM Tris, pH 7.4, 100 mM NaCl, with 1× protease inhibitor cocktail, and 1× phosphatase inhibitor cocktail (Sigma, St. Louis, MO, USA). Lysates were centrifuged at 13,000g for 10 min at 4 °C to precipitate the debris, and the protein content in the supernatant determined using the Bio-Rad protein assay (Bio-Rad Laboratories, CA, USA). Lysate protein (20 µg/lane) was separated using 4–20% gradient gels (Bio-Red, Hercules, CA, USA) and transferred to PVDF membranes with the Trans-Blot® Turbo™ Transfer System(Bio-Red, Hercules, CA, USA). The blots were then probed with the appropriate antibody overnight at 4 °C. Primary antibodies used were anti-IRP-1 (Abcam Inc., Cambridge, MA, USA); anti-Ferritin (Abcam Inc., Cambridge, MA, USA); anti-TfR (Abcam Inc., Cambridge, MA, USA). Blots were washed in 1× TBST (3×15 min) and the appropriate secondary antibodies conjugated to HRP (Sigma, St. Louis, MO, USA) were then added for 1 h at RT (Thermo Scientific, Rockford, IL, USA). After further washing in TBST (3×15 min) bands were visualized by chemiluminescence (West-Femto, Pierce, Rockford, IL, USA) and quantified using a Kodak Molecular Imaging System (Kodak, Rochester, NY, USA).
9
Spinal Cord Protein Analysis in Mice
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After 6 weeks, the mice were euthanized with pentobarbital. The spinal cords were dissected and homogenized in RIPA buffer (50 mM Tris-Cl, pH 7.4, 1% NP-40, 0.1% sodium dodecyl sulfate [SDS], and 150 mM NaCl) containing a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL, USA). The homogenized tissue was centrifuged at 19.083 g for 20 min at 4°C. Total protein was quantified using the bicinchoninic acid assay kit (Pierce Biotechnology Inc., Rockford, IL, USA). Samples denatured in SDS sampling buffer were separated by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA) for Western blotting. For detection of target proteins, the membranes were blocked with 5% skim milk (Sigma-Aldrich, St. Louis, MO, USA) in Tris-buffered saline and incubated with various primary antibodies, including anti-tubulin, anti-HO1, anti-ferritin, and anti-TNF-α (Abcam, Cambridge, UK; 1:1000), anti-Iba-1 (Wako, Osaka, Japan; 1:1000), and anti-TLR4, anti-transferrin, anti-BAX, and anti-NQO1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; 1:1000). Subsequently, the blots were probed with horseradish peroxidase-conjugated antibodies (Santa Cruz Biotechnology) and visualized using Femto (Thermo Fisher Scientific). A ChemiDoc image analyzer (Bio-Rad) was used to detect immunoblotted bands.
10
Puerarin Attenuates LPS-Induced Oxidative Stress
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48 healthy male Sprague-Dawley rats (aged 8-10 weeks) were purchased from Model Animal Research Center. Puerarin was obtained from Sigma-Aldrich (P5555). Lipopolysaccharide (LPS, ST1470) and One Step TUNEL Apoptosis Assay Kit (C1088) were purchased from Beyotime Biotechnology. The following antibodies were used in this study: anti-cleaved caspase3 (Cell Signaling Technology, 9661), anti-Bax (Cell Signaling Technology, 2772), anti-Bcl2 (Cell Signaling Technology, 3498), anti-GPX4 (Cell Signaling Technology, 52455), anti-ACSL4 (Santa Cruz Biotechnology, sc-365230), anti-Ferritin (Abcam, ab75973), anti-TFR (Abcam, ab84036), anti-P-AMPK (Cell Signaling Technology, 50081), anti-T-AMPK (Cell Signaling Technology, 5832), and anti-GADPH (Beyotime Biotechnology, AF0006). Then enzyme-linked immunosorbent assay (ELISA) kit for the detection of IL-6 (H007), IL-10 (H009) and TNF-α (H052) were purchased from Nanjing Jiancheng Bioengineering Institute. In addition, oxidative stress indicators (MDA, GSH, and SOD) were detected by the ELISA kit from Nanjing Jiancheng Bioengineering Institute.
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