Samples were loaded onto the pre-wetted nitrocellulose membrane using Bio-Dot microfiltration apparatus (Bio-rad). After washing each sample with tris-buffered saline, samples were blocked with 5% non-fat dry milk in tris-buffered saline containing 0.1% tween-20. Membranes were incubated with anti-α-synuclein filament antibody (1:1000; Abcam) or GlcCer antibody (1:500, Glycobiotech) at 4 °C overnight, followed by HRP-conjugated rabbit secondary antibody (GE Healthcare) for 1 h at RT.
Hrp conjugated rabbit secondary antibody
Manufactured by GE Healthcare
Sourced in United States
The HRP-conjugated rabbit secondary antibody is a reagent used in immunoassay and immunohistochemistry applications. It functions as a detection tool, binding to primary antibodies raised in rabbits and enabling the visualization of target analytes or antigens through an enzymatic reaction.
Automatically generated - may contain errors
Lab products found in correlation
Bio dot microfiltration apparatus, by Bio-Rad (2 mentions)
P mlc2, by Cell Signaling Technology (2 mentions)
Tenascin c, by Abcam (2 mentions)
Total fak, by BD (2 mentions)
Total mlc2, by Abcam (2 mentions)
Alexa fluor conjugated goat secondary anti mouse igg and anti rabbit igg antibodies, by Thermo Fisher Scientific (2 mentions)
Na934v, by GE Healthcare (2 mentions)
Py397 fak, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Lc3a b, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Phosphoserine antibody, by Merck Group (1 mentions)
Bis tris plus gel, by Thermo Fisher Scientific (1 mentions)
Nupage lds sample buffer 4x, by Thermo Fisher Scientific (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Hif 2α, by BD (1 mentions)
9 protocols using hrp conjugated rabbit secondary antibody
1
Immunoblotting of α-Synuclein and GlcCer
2
Antibody Validation for Cellular Analysis
3
Western Blot Analysis of Cellular Proteins
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Cell lysates were prepared in Laemmli Sample Buffer (BIO‐RAD, Hercules, CA, USA) containing 2‐mercaptoethanol and heated for 5 min at 95°C. Cell lysates were separated in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% skim milk in T‐PBS for 60 min at room temperature. Then the membrane was incubated with the appropriate antibodies overnight. The membrane was washed three times with T‐PBS and incubated with HRP‐conjugated goat anti‐rabbit antibodies for 30 min, and specific proteins were detected using an enhanced chemiluminescence detection system.
Primary antibodies were obtained from Cell Signaling Technology Tokyo, Japan (HMOX1[HO‐1], C‐PARP, LC3A/B, β‐actin). HRP‐conjugated rabbit secondary antibody was purchased from GE Healthcare Life Sciences (Piscataway, NJ, USA)
Primary antibodies were obtained from Cell Signaling Technology Tokyo, Japan (HMOX1[HO‐1], C‐PARP, LC3A/B, β‐actin). HRP‐conjugated rabbit secondary antibody was purchased from GE Healthcare Life Sciences (Piscataway, NJ, USA)
4
Dot Blot Assay for Oligomeric A11 Detection
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Samples were placed into the pre-wetted nitrocellulose membrane in the Bio-Dot microfiltration apparatus (Bio-rad). Under vacuum, 20 μg of tissue lysates were loaded onto the membrane. Following a Tris-buffered saline wash, samples were blocked in a solution that contained 0.1% Tween-20 and 5% non-fat dry milk. Membranes were incubated with anti-A11 oligomers antibody (1:500, Life technologies) at 4°C overnight, followed by HRP-conjugated rabbit secondary antibody (1:50,000, GE Healthcare) for 1 h at room temperature and the signals were visualized using chemiluminescence reagents (Thermo Scientific).
5
Akt Isoforms Phosphorylate Cytochrome c
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Recombinant Akt 1 (#A16-10G), Akt 2 (#A17-10G), and Akt 3 (#A18-10G) isoforms were purchased from SignalChem (Richmond, British Columbia, Canada). The in vitro kinase reaction was performed in 50 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 200 µM ATP, 0.01% Brij-35 buffer with 2 µM Cytc at 30 °C. The serine phosphorylation state of Cytc was determined by Western blotting at times 0, 15, and 30 min. The blots were probed with a 1:5000 dilution of phosphoserine antibody (Millipore, Burlington, MA, USA #AB1603) in 5% BSA-TBST buffer, followed by a 1:10,000 dilution of HRP-conjugated rabbit secondary antibody (GE Healthcare).
6
Antibody Validation for Cellular Analysis
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Antibodies were as follows (used at 1μg ml−1 for western blotting and chromatin immunoprecipitation (ChIP) and at indicated concentrations for immunofluorescence studies): tenascin C (Abcam ab108930; 1:1,000), pY397-FAK (Invitrogen 44625; 1:200), total FAK (BD Biosciences 610088), pMLC2 (Cell Signaling 3671; 1:200), total MLC2 (Abcam ab92721, clone EPR3741; 1:200), p-MyPT1 (Millipore ABS45; 1:200), hyaluronic acid binding protein (Calbiochem, 385911; 1:500), aggrecan (Abcam ab3778; 1:500), versican (Abcam ab19345; 1:500), collagen 1 (Abcam ab34710; 1:1,000), propidium iodide (AcrosOrganics 440300250; 1 μg ml−1), β-actin (Sigma-Aldrich a5441), HIF1α (Abcam ab-1, for immunofluorescence; 1:200; Abcam ab1, for ChIP; Novus 100-449, for western blotting), hypoxyprobe (Hypoxyprobe, HP1-100 Kit; per manual), CD31 (BD 550389; 1:500), laminin (Abcam ab11575; 1:500), RNA polymerase II (Millipore 05-623B), rabbit IgG isotype control (Cell Signaling 2729; 1:500), Alexa Fluor-conjugated goat secondary anti-mouse IgG and anti-rabbit IgG antibodies (Invitrogen A11012 and A11005; 1:500) and HRP-conjugated rabbit secondary antibody (GE Healthcare Life Sciences NA934VS; 1:5,000).
7
Immunoblotting Analysis of SNAP-Tag Proteins
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Cells were lysed in RIPA lysis buffer (50 mM Tris HCl pH 7.4, 1% Triton X-100, 0.5% Sodium Deoxycholate, 0.1% SDS, 50 mM NaF, 120 mM NaCl, 5 mM β-glycerophosphate) supplemented with protease and phosphatase inhibitors (1 mM benzamidine, 1 mM AEBSF, 2 µg/mL leupeptin, 100 nM microcystin-LR). Lysed samples were boiled for 5 min at 95°C in NuPAGE LDS Sample Buffer 4X (Thermo Fisher) + 5% BME (Sigma-Aldrich). Protein concentration was determined using a Pierce BCA Protein Assay Kit (Thermo Fisher). Samples were resolved on Bolt 4–12% Bis-Tris Plus Gels (Invitrogen) or AnykD Criterion TGX Precast Midi Protein Gel (Biorad). Proteins were transferred to nitrocellulose for immunoblotting and probed with Anti-SNAP-tag rabbit antibody (NEB) and Anti-GAPDH−HRP mouse mAb, (Novus). Detection was achieved with a HRP-conjugated rabbit secondary antibody (GE Healthcare) followed by enhanced chemiluminescence with SuperSignal West Dura Extended Duration Substrate (Thermo Fisher). Densitometry was performed using NIH ImageJ (Fiji) software.
8
Western Blot Analysis of Hypoxia Signaling
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Whole cell lysates were prepared in modified RIPA buffer and proteins were fractionated by SDS-PAGE. The following primary antibodies were used: HIF-1α (BD Biosciences); HIF-2α, SLC1A1, SLC1A3, GRIA2, GRIA3, FYN, pLYN (Tyr396), LYN, SRC, LCK, ERK, Caspase-3, and AKT (Novus Biologicals); and phosphorylated ERK, phosphorylated AKT (Ser473), and β-actin (Santa Cruz). HRP-conjugated secondary rabbit antibody was purchased from GE Healthcare Life Sciences; all other secondary antibodies were obtained from Santa Cruz.
9
Histone and Whole-Cell Lysate Analysis
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Cells were trypsinized for histone extraction as per the Abcam protocol. Additionally, cells were lysed using RIPA buffer for whole-cell lysates. Protein concentration was assessed (BioRad, 5000116). A total of 10 µg total histones and 40 µg whole-cell lysates were loaded on gels. Membranes were incubated overnight at 4 °C with primary antibody in 5% BSA in TBST. Primary antibodies used were: H3K27me3 (Cell signaling, 9733S), total histone H3 (Cell Signaling, 9715), EZH2 (Cell signaling, 5246), KDM6A (Atlas Antibodies, HPA002111), ALDH2 (Abcam, ab108306), and CK5 (Covance, PRB 160-P). Membranes were incubated with HRP-conjugated secondary rabbit antibody (GE Life Sciences, NA934V). The secondary antibody was detected using Luminata Crescendo Western HRP Substrate (WBLUC0500). The membranes were stripped with Restore Plus (Thermo Scientific, 46428) and re-probed with GAPDH (Abcam, ab9485) or Total H3.
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