Protein lysates were denatured and separated on 10% polyacrylamide gels. After transfer to polyvinylidene fluoride (PVDF) membranes (Roche Applied Science), membranes were blocked in 5% nonfat milk powder for one hour at room temperature and incubated with primary antibodies overnight at 4 °C. After several times of wash with TBST, membranes were incubated with secondary antibody for one hour at room temperature. Finally, the membranes were visualized using the Tanon 5200 Western blotting Detection System (Tanon, Shanghai, China). Primary antibodies used were anti-ETV6 (ThermoFisher Scientific, Waltham, MA, USA; PA5-35371, 1:1000), anti-AKT (Cell Signaling Technology, Danvers, MA, USA; 4691T, 1:1000), anti-p-AKT (Cell Signaling Technology, Danvers, MA, USA; 4060T, 1:1000), anti-p-MEK1/2 (Cell Signaling Technology, Danvers, MA, USA; 9154, 1:1000), and anti-GAPDH (Weiao, Shanghai, China; WB0197, 1:1000). For secondary antibodies, anti-rabbit IgG (Abcam, Cambridge, UK; 7074, 1:10000), anti-mouse IgG (Abcam, Cambridge, UK; 7076, 1:10000), and HRP anti-rabbit (Santa Cruz Biotech., Santa Cruz, CA, USA; 1:10000) were used. Signal was enhanced using an enhanced chemiluminescence technique (Thermo Scientific, Waltham, MA, USA; 34075).
Anti rabbit hrp
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-rabbit-HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and bind to primary antibodies raised in rabbits, allowing for amplification and visualization of the target antigen in various immunoassays and immunohistochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse hrp, by Santa Cruz Biotechnology (15 mentions)
Anti goat hrp, by Santa Cruz Biotechnology (5 mentions)
Western lightning plus ecl, by PerkinElmer (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Anti p akt, by Cell Signaling Technology (3 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti erk1 2, by Cell Signaling Technology (2 mentions)
Anti p erk1 2, by Cell Signaling Technology (2 mentions)
Gapdh, by Abcam (2 mentions)
Anti nanoluc, by Promega (2 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Criterion xt bis tris protein gel, by Bio-Rad (2 mentions)
Anti ftsz, by Merck Group (2 mentions)
Anti mouse hrp, by Thermo Fisher Scientific (2 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Anti actin, by Santa Cruz Biotechnology (2 mentions)
Chemiluminescence reagent, by PerkinElmer (2 mentions)
Ab175402, by Abcam (2 mentions)
47 protocols using anti rabbit hrp
1
Western Blot Analysis of Protein Expression
2
EGCG Quantification and Protein Analysis
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The purified EGCG (95%) was purchased from the commercial herb supplier, Maysar Herbals (Haryana, India). The primary antibodies specific for ALCAM (#sc-74557), CD31 (#sc-376764), β-actin(#sc-47778), Histone H3 (#sc-5661), and secondary antibodies horseradish peroxidase(HRP)-linked anti-mouse (#sc-2031), HRP-anti-rabbit (#sc-2030) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), TFAP-2A (#HPA056871) and TFAP-2C(#SAB2102408) primary antibodies were purchased from Sigma-Aldrich (St. Louis, MO). Anti-miR-214 inhibitor (#4464084), lipofectamine (#11668019), and custom primers specific for miR-214 and U6 were obtained from Invitrogen (Carlsbad, CA). All antibodies and other chemicals were purchased through an Indian supplier (New Delhi, India). The cell culture mediums and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific India Pvt. Ltd., Mumbai, India.
3
Western Blot Analysis of Autophagy Markers
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293 T cells were lysed in NP40 lysis buffer (50 mM Tris–HCl pH8, 0.5% NP40, 0.1% EDTA, 10% glycerol and protease inhibitors), mixed with 2× SDS sample buffer and loaded onto 10% PAGE pre-cast gels (BioRad). Proteins were transferred to a PVDF membrane and subsequently blocked for 1 to 2 h in blocking solution (5% skimmed milk and 0.1% Tween in PBS). Proteins of interest were probed with primary antibodies overnight at 4°C in blocking solution. HRP-conjugated secondary antibody incubation was conducted for 1 h at room temperature in blocking solution. Antibody bound proteins were detected using ECL reagents. The following antibodies were used: anti-GLUC (Ketteler et al. [19 (link),20 (link)], anti-Flag M2 (Sigma-Aldrich), anti-β-Actin (Abcam), anti-LC3 (Sigma-Aldrich), anti-ATG5 (Nanotools), HRP anti-mouse (Santa-Cruz) and HRP anti-rabbit (Santa-Cruz).
4
Western Blot Analysis of Signaling Proteins
5
Western Blot Analysis of Drosophila Tissues
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Wing imaginal discs were dissected in chilled PBS supplemented with protease inhibitor (#11697498001; Roche) and directly transferred into Laemmli sample buffer, then boiled for 10 min. Hemolymph was extracted by bleeding larvae into chilled PBS supplemented with protease inhibitor on a cold aluminum block using a fine tungsten needle to puncture the cuticle, then transferred into Laemmli sample buffer and boiled for 10 min. Samples were run on 10% Mini-Protean TGX gels (Bio-Rad) and transferred to nitrocellulose membrane (Bio-Rad). The primary antibody used was rabbit anti-GFP (#TP401, 1:1,000; Torrey Pines Biolabs). Protein bands were detected with secondary antibody HRP anti-rabbit (#sc-2030, 1:2,500; Santa Cruz Biotechnology), and Western Lightning Plus-ECL (#NEL103001EA; PerkinElmer).
6
Macrophage Immunological Profiling
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The following antibodies and conjugates were used in this study: anti‐F4/80 clone Cl:A3‐1. LXRα/β antiserum,[43 (link)
] LXRα (Abcam#PPZ0412), H3K27Ac (Abcam#ab4729), NOS‐2 (Santa Cruz#SC‐650 M‐19), ABCA1 (Novus Biologicals #NB400‐105), GAPDH (Abcam #ab9485‐100), Anti‐mouse‐HRP (Santa Cruz#SC‐2005), Anti‐rabbit‐HRP (Santa Cruz#SC‐2004), CD11b‐PerCP‐Cy5.5 (Biolegend #clone M1/70), F4/80‐PE or FITC (Ebioscience #clone BM8), Ly6G‐PE or FITC (BD Pharmingen #clone AL21), MHC‐II‐APC (eBioscience #M5/114.15.2). The following pharmacological reagents were obtained from the MRC PPU Reagents University of Dundee, UK: BI605906 (IKKβ inhibitor), 5Z‐7‐oxozeanol (TAK1 inhibitor), MRT67307 (TBK1 inhibitor) and the following products from Calbiochem: PD0325901 (MEK/ERK inhibitor), PD98059 (MEK1 and MEK2 inhibitor), PIK‐75‐hydrochloride (inhibitor of p110α subunit of PI3‐Kinase), SB590885 (Raf‐1 inhibitor); HX531 (RXR inhibitor). TLR agonists, Poly I:C (TLR3 agonist) and ultrapure LPS from E. coli 0111:B4 strain‐ (TLR4 ligand) were obtained from Invivogen and were used at 10 µg ml−1 and 100 ng ml−1 respectively.
] LXRα (Abcam#PPZ0412), H3K27Ac (Abcam#ab4729), NOS‐2 (Santa Cruz#SC‐650 M‐19), ABCA1 (Novus Biologicals #NB400‐105), GAPDH (Abcam #ab9485‐100), Anti‐mouse‐HRP (Santa Cruz#SC‐2005), Anti‐rabbit‐HRP (Santa Cruz#SC‐2004), CD11b‐PerCP‐Cy5.5 (Biolegend #clone M1/70), F4/80‐PE or FITC (Ebioscience #clone BM8), Ly6G‐PE or FITC (BD Pharmingen #clone AL21), MHC‐II‐APC (eBioscience #M5/114.15.2). The following pharmacological reagents were obtained from the MRC PPU Reagents University of Dundee, UK: BI605906 (IKKβ inhibitor), 5Z‐7‐oxozeanol (TAK1 inhibitor), MRT67307 (TBK1 inhibitor) and the following products from Calbiochem: PD0325901 (MEK/ERK inhibitor), PD98059 (MEK1 and MEK2 inhibitor), PIK‐75‐hydrochloride (inhibitor of p110α subunit of PI3‐Kinase), SB590885 (Raf‐1 inhibitor); HX531 (RXR inhibitor). TLR agonists, Poly I:C (TLR3 agonist) and ultrapure LPS from E. coli 0111:B4 strain‐ (TLR4 ligand) were obtained from Invivogen and were used at 10 µg ml−1 and 100 ng ml−1 respectively.
7
Western Blot Analysis of Bacterial Proteins
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Cells were grown in LB with appropriate antibiotics to OD600 ~ 1. 1 mL of culture was harvested by centrifugation, resuspended in lysis buffer (100 mM NaCl, 50 mM EDTA) with 7 μl of 10 mg ml−1 lysozyme and incubated at 37 °C for 30 min. After 40 μl of 20% sarkosyl was added, the samples were incubated for 5 min at 37 °C. Then, 6X loading dye (250 mM Tris pH 6.8, 20% glycerol, 30% β-mercaptoethanol, 10% SDS, saturated bromophenol blue) was added and the lysate was denatured at 90 °C for 10 min. Protein was separated on either a 4–12% or 12% Criterion XT Bis-Tris protein gel (Bio-Rad) with XT-MES buffer and transferred to polyvinylidene membranes using the Trans-Blot Turbo Transfer system (Bio-Rad). Membranes were blocked in 5% milk for 1 h at room temperature, washed and then probed with antibodies diluted in TBS-Tween at the following dilutions: anti-NanoLuc, 1:2,000 (Promega); anti-FtsZ, 1:2,000 (Sigma); anti-mouse-HRP, 1:4,000 (Thermo Fisher); anti-rabbit-HRP, 1:4,000 (Santa Cruz Biotechnologies). Chemiluminescent signals from HRP were detected using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher) and were visualized using the ChemiDoc Imaging System (Bio-Rad).
8
Western Blot Analysis of CSF Proteins
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CSF samples obtained from embryonic lateral ventricles were lysed in RIPA buffer (Thermo Fisher Scientific, USA) containing Halt protease inhibitor cocktail (Thermo Fisher Scientific, USA). The lysates were separated on 4%–20% SDS polyacrylamide gels and blotted on Immobilon‐P PVDF membranes (Merck Millipore, USA). For Western blot detection, the PVDF membranes were treated with anti‐Pdgf‐A (Abbiotec, USA), anti‐Wnt3a (Cell Signaling Technology, USA) or anti‐alpha‐Tubulin‐HRP (Proteintech, USA) primary antibodies in Tris‐buffered saline with 0.1% Tween 20 and 5% skim milk. For Pdgf‐A detection, anti‐rabbit‐HRP (Santa Cruz Biotechnology, USA) secondary antibodies were used, and HRP signals were visualized with an Enhanced Chemiluminescence (ECL) Reagent Kit (BD Pharmingen, USA).
9
Western Blot Analysis of Protein Expression
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Cellular extracts were prepared using lysis buffer containing 50 mM Tris HCL (pH 7.5), 250 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, and EDTA free protease inhibitor (Roche 11873580001). Extracts were run on a 4–12% Tris-Glycine gel (BioRad) and transferred onto PVDF membranes. Blots were blocked in 5% milk PBS-T for 1 h at room temperature followed by overnight incubation at 4 °C with primary antibodies at 1:1,000 (anti-HA Tag, Cell Signaling 3724S; anti-Flag Tag, Sigma F1804; anti-DDX5, Bethyl A300-523A; anti-DDX17, Bethyl A300-509A). Horseradish peroxidase (HRP) -conjugated secondary antibodies were used at 1:10,000 (anti-rabbit HRP, Santa Cruz sc-2030) or 1:1,000 (anti-mouse HRP, Cell Signaling 7076S).
10
Quantitative Western Blot Analysis of Pathogen-Induced Proteins
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Equal amounts of protein were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Sigma-Aldrich, St. Louis, MO, USA) with Mini-PROTEAN® Tetra Handcast Systems (Bio-Rad, Hercules, CA, USA). The membranes were incubated with primary antibodies (for anti-GFP (Abcam, AB6556), 1:5000; anti-HA (Roche, 11867423001), 1:5000; anti-PR1, 1:5000; anti-RbCL (Clontech 632475), 1:2000; anti-GST (Virogen, 101-A-100), 1:5000; or anti-His (Thermo Fisher, PA1-23024), 1:5000) in 1× TBS with 5% w/v skim milk at 4 °C overnight. Treated membranes were washed with 1× TBS and incubated with a secondary horse radish peroxidase (HRP)-conjugated antibody (anti-rabbit HRP (Santa Cruz, sc-2004), 1:10,000; anti-rat (Santa Cruz, sc-2006), 1:10,000; anti-mouse (GE Healthcare Life Science; NA931), 1:10,000) at room temperature for 2 h. Signals were visualized using the Pierce ECL and ChemiDoc MP (Bio-Rad) systems. Protein band intensity was measured by using ImageJ software. Relative intensity protein band under Pto infection condition was calculated by normalizing to the intensity of protein band under mock condition, which was set as 1.
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