Proteins extracted from cell lysate were separated with SDS-PAGE (10% polyacrylamide gels) and then transferred onto the polyvinylidine difluoride (PVDF) membranes. The membranes were blocked by 5% skimmed milk (room temperature; 1 h) and then incubated with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (ab8245, Abcam, 1:10,000) and mouse Siglec-1 antibody (#MAB5610, R&D Systems, 1 μg/mL) (4°C; overnight); the membrane was incubated with anti-rat immunoglobulin G (IgG), horseradish peroxidase (HRP)-linked antibody (#7077, Cell Signaling Technology, 1:1,000) at room temperature (1 h). After rinsing with PBST (PBS with 0.1% Tween-20) five times, positive signals were detected using SuperSignalWest Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific, USA) and scanned using a Tanon 4200SF chemiluminescent imaging system (Tanon, Shanghai, China).
Horseradish peroxidase hrp linked antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Horseradish peroxidase (HRP)-linked antibody is a laboratory reagent that serves as a detection agent. HRP is an enzyme that can catalyze a color-producing reaction, allowing the detection and visualization of target proteins in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Ab8245, by Abcam (1 mentions)
Ccr5 antibody, by Santa Cruz Biotechnology (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Quercetin, by Beyotime (1 mentions)
Anti collagen 4, by Abcam (1 mentions)
Reactive oxygen species assay kit, by Yeasen (1 mentions)
Gapdh d16h11 xp rabbit mab, by Cell Signaling Technology (1 mentions)
Hrp linked antibody, by Cell Signaling Technology (1 mentions)
Erastin hy 15763, by MedChemExpress (1 mentions)
Calcein pi live dead viability cytotoxicity assay kit, by Beyotime (1 mentions)
Quercetin, by Merck Group (1 mentions)
Ferrostatin 1, by Merck Group (1 mentions)
Cell cycle and apoptosis analysis kit, by Beyotime (1 mentions)
Mitochondrial membrane potential assay kit with jc 1, by Beyotime (1 mentions)
Cell counting kit 8 cck 8 kit, by Beyotime (1 mentions)
Fluorescent secondary antibody, by LI COR (1 mentions)
Pdgfrβ, by Cell Signaling Technology (1 mentions)
Mmp 9, by Abcam (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
9 protocols using horseradish peroxidase hrp linked antibody
1
Western Blot Analysis of Protein Expression
2
Quantitative Western Blot Analysis
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Western blotting, immunoblotting, and densitometry were performed as described earlier [17 (link)]. We used the biorad ChemiDoc XRT+ device. The antibodies used to detect and quantify the antigens are listed in Table 3 . The applied secondary antibody, a Horseradish peroxidase (HRP)-linked antibody was utilized at a dilution of 1:4000 (Cell Signaling Technology, Inc., Danvers, MA, USA). In addition, we used glyceraldehyde 3-phosphate dehydrogenase (GAPDH; dilution: 1:1000). Ponceau S red staining was used as an alternative to housekeeping proteins as loading controls. The membranes were analyzed using ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih.gov/ij/ ), for densitrometric quantification of the bands. Ponceau S was evaluated according to [51 ]. Statistical analyses were performed as previously published [17 (link)].
3
CCL11-CCR5 Interaction Quantification
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Recombinant CCL11 was coated onto the wells of maxisorp 96-well microtiter plates and incubated for 18 hours at 4°C. Each well was washed three times with PBST (0.2% Tween-20 in PBS). Thereafter, the plates were blocked with 2% BSA in PBST for 2 hours. Cell lysates were added to the plates and incubated for 2 hours. The wells were then washed three times [27 (link)]. Preparations of the CCR5 antibody (Santa Cruz Biotechnology) in blocking solution were added to the plates and allowed to react for 2 hours. After washing, horseradish peroxidase (HRP)-linked antibody (Cell signaling Technology) was added and the lysates were incubated for 2 hours; this was followed by five rounds of washes. The reaction was developed with 100 μL tetramethylbenzidine (TMB) substrate solution and stopped with 100 μL of 1 N H2SO4. The absorbance in the microtiter plates was measured at 450 nm using a microplate reader (Infinite 200 PRO; Tecan Life Sciences, Zürich, Switzerland).
4
Quercetin, Erastin, and Ferrostatin-1 Protocol
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Quercetin (Q4951, purity > 95%, Sigma-Aldrich), erastin (HY-15763, purity > 99% MedChemExpress) and ferrostatin-1 (SML0583, purity > 95%, Sigma-Aldrich) were dissolved in DMSO. For the cell experiments, Quercetin was dissolved in DMSO to achieve the pre-designed concentration and cells were treated with Quercetin for 24 h or 48 h. The following kits were used in the present study: Cell-Counting-Kit-8 (CCK-8) kit, DAPI Staining Solution, Crystal Violet Staining Solution, Calcein/PI Live/Dead Viability/Cytotoxicity Assay kit, Cell Cycle and Apoptosis Analysis kit, Cell Cycle and Apoptosis Analysis kit, mitochondrial membrane potential assay kit with JC-1 (Beyotime, China); Reactive Oxygen Species Assay kit (YEASEN, China). The antibodies used in this study included primary antibodies to anti-p53, anti-xCT, anti-glutathione peroxidase 4, anti-aconitase 1 (ACO1), anti-transferrin receptor, anti-ferritin light chain and anti-collagen IV (Abcam, USA); GAPDH (D16H11) XP rabbit mAb, anti-rabbit IgG, horse radish peroxidase (HRP)-linked antibody, anti-mouse IgG, and HRP-linked antibody (Cell Signaling Technology, USA).
5
Immunoblotting Antibody Panel for Apoptosis
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Bcl-2 (D17C4) rabbit monoclonal antibody (mAb), Bax (D2E11) rabbit mAb, caspase-3 rabbit mAb, cleaved caspase-3 (Asp175) antibody, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (D16H11) XP rabbit mAb, β-Tubulin (9F3) rabbit mAb, PI3K p110a (C73F8) rabbit mAb, Akt (pan) (11E7) rabbit mAb, phospho-Akt (Ser473) (D9e) XP rabbit mAb and anti-rabbit IgG, Horseradish Peroxidase (HRP)-linked antibody was purchased from Cell Signalling Technology (Boston, Massachusetts, USA) were obtained for these studies. Fluorescent secondary antibodies were purchased from LI-COR Biosciences (Lincoln, Nebraska, United States).
6
FLP Ointment and Celecoxib Protocol
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FLP ointment (No. Z20063236) was prepared by Pharmaceutical preparation center of Guang’anmen Hospital, China Academy of Chinese Medical Sciences. Celecoxib (No. H20120355) was purchased from Pfizer Pharmaceuticals LLC (Dalian, China). Antibodies against Cox-2, E-Cadherin, Vimentin, platelet-derived growth factor receptors (PDGFR) β, β-actin, GAPDH, anti-rabbit IgG, and horseradish peroxidase (HRP)-linked antibody was purchased from Cell Signaling Technology (Cambridge, MA, USA). Vascular endothelial growth factor (VEGF), N-Cadherin, microsomal Prostaglandin E synthase-1 (mPGES-1), matrix metalloproteinases (MMP)-2, and MMP-9 antibodies were purchased from Abcam (Cambridge, MA, USA). Biotinylated secondary antibodies were purchased from Beijing Xiya Jinqiao Biological Technology Co. Ltd.
7
Lipid and Protein Extraction Protocol
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Palmitic acid (PA, sodium salt) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA, sodium salt) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Palmitic acid was dissolved in 50% ethanol, while docosahexaenoic acid was dissolved in 100% ethanol to a concentration of 10 mg/mL; these stock solutions were stored at −80 °C under N2 until use. The rabbit monoclonal anti-Calnexin (C5C9) and the rabbit monoclonal anti-Histone H3 (D1H2) antibodies were purchased from Cell Signaling Technology, Danvers, MA, USA. The mouse monoclonal anti-HSP60 (66041) antibody was purchased from Proteintech, Deansgate, Manchester, UK. Bound primary antibody was visualized by proper secondary horseradish peroxidase (HRP)-linked antibody, purchased from Cell Signaling Technology, Danvers, MA, USA. All solvents were purchased from Carlo Erba Reagents, Italy. Avanti Polar Lipids Inc. (Alabaster, AL, USA) supplied the fatty acid methyl ester (FAME) standards for GC analysis and neutral lipids (TG and CE) for LC analysis. Phospholipid standards were purchased from Sigma Aldrich, St. Louis, MO, USA.
8
Comprehensive Protein Quantification Protocol
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The primary antibodies used in this study include rabbit anti-MyoVa (dilute 1:500 for immunofluorescence and 1:1000 for Western blotting; Sigma-Aldrich, HPA001356), mouse anti-MICAL1 (dilute 1:1000 for Western blotting; Sigma-Aldrich, HPA040902), rabbit anti-MICAL1 (dilute 1:200 for immunofluorescence; ProteinTech, 14818-1-AP), mouse anti–β-tubulin (dilute 1:1000 for immunofluorescence; Sangon, D1930693), mouse anti–Aurora B (dilute 1:500 for immunofluorescence; BD, 611082), mouse anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (dilute 1:1000 for Western blotting; Transgene, HT801-01), and mouse anti-Flag (dilute 1:1000 for Western blotting; Sigma-Aldrich, F1804) antibodies. The secondary antibodies used in this study include gat anti-mouse immunoglobulin G (IgG), horseradish peroxidase (HRP)–linked antibody [dilute 1:3000; Cell Signaling Technology (CST)], goat anti-rabbit IgG, HRP-linked antibody (dilute 1:3000; CST) for Western blotting, and Alexa Fluor 488/594/633–conjugated anti-mouse/rabbit IgG antibody (dilute 1:3000; Invitrogen) for immunofluorescence experiments.
9
Western Blot Analysis of Myostatin, Oct4, and PCNA
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Immuno-detection on the membranes was with primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) against myostatin (GDF8/11, mouse monoclonal antibody SC-393335); and housekeeping beta-actin (mouse monoclonal, followed by secondary antibodies: anti-mouse IgG, horseradish peroxidase (HRP)-linked antibody (Cell Signaling Technology, Danvers, MA, USA), or anti-rabbit IgG linked to HRP (Amersham GE, Pittsburgh, PA, USA). Oct4 was estimated by a polyclonal antibody from Biovision catalog 3576, at 1:500; and PCNA with a monoclonal antibody from Millipore MAB424, at 1:2000. Bands were visualized using luminol (SuperSignal West Pico; Chemiluminescent, Pierce, Rockford, IL, USA). For negative controls, the primary antibody was omitted. Densitometric analysis was performed in certain cases, as stated, correcting by the housekeeping proteins [5 (link),7 (link),8 (link)].
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