Copper dichloride (CuCl2) was purchased from Sangon Biotech (Shang Hai, China). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (CCK-8) was purchased from Sigma-Aldrich (China). The primary antibody for phospho-EGFR (Y1068, #4064) and phospho-AKT (S473, #4007) was obtained from Bioworld Technology. The primary antibody for phospho-MET (Y1234/Y1235, #3077), total-MET (#8198), and phospho-ERK (T202/Y204, #3510) was obtained from Cell Signaling Technology. The secondary antibody (goat anti-rabbit IgG(H&L)-HRP, goat anti-mouse IgG(H&L)-HRP) was obtained from Invitrogen. The α-tubulin primary antibody was purchased from Cellway Biological Co., Ltd. Recombinant Human Hepatocyte Growth Factor (hHGF) and Recombinant Human Epidermal growth factor (hEGF) were obtained from Peprotech. Forenitib, a MET inhibitor, was purchased from Selleck. The nitrocellulose membrane was obtained from Merck Millipore (Germany). RPMI1640, DMEM medium was purchased from neuronbc (Beijing, China); fetal bovine serum (FBS) was purchased from Biological Industries USA. Penicillin-Streptomycin (100X), 0.25% Trypsin-EDTA (1X), and ECL substrate solution were purchased from NCM Biotech (Suzhou, China).
Goat anti rabbit igg h l hrp
Manufactured by Thermo Fisher Scientific
Sourced in United States
Goat anti-rabbit IgG H&L (HRP) is a secondary antibody conjugated with horseradish peroxidase (HRP). It is used to detect and quantify rabbit primary antibodies in various immunoassays and immunochemical techniques.
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Lab products found in correlation
Goat anti mouse igg h l hrp, by Thermo Fisher Scientific (17 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (3 mentions)
Goat anti mouse igg hrp, by Thermo Fisher Scientific (2 mentions)
Mouse monoclonal anti vp16 antibody, by Santa Cruz Biotechnology (2 mentions)
Mouse anti β actin monoclonal antibody, by Sino Biological (2 mentions)
Mouse monoclonal anti icp0 antibody, by Santa Cruz Biotechnology (2 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
α brdu, by BD (2 mentions)
Normal mouse igg, by Merck Group (2 mentions)
α macroh2a1, by Merck Group (2 mentions)
α h2ax, by Abcam (2 mentions)
α h2b, by Abcam (2 mentions)
α brca1, by Santa Cruz Biotechnology (2 mentions)
α gapdh, by Santa Cruz Biotechnology (2 mentions)
α γh2ax, by Merck Group (2 mentions)
α phospho s139 h2ax, by Merck Group (2 mentions)
α h2a, by Abcam (2 mentions)
Img 124a, by Merck Group (2 mentions)
34 protocols using goat anti rabbit igg h l hrp
1
MET and EGFR Signaling Pathway Inhibitors
2
Anti-inflammatory effects of LPS and λ-carrageenan
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Escherichia coli LPS, and λ-carrageenan were obtained from Sigma (St. Louis, MO, USA). Primary antibodies (Anti-iNOS, anti-COX2, anti-IκBα, anti-p-IκBα, anti-β-actin antibodies) and secondary antibody (goat anti-rabbit IgG (H + L), HRP) were purchased from Invitrogen (Rockford, IL, USA). Anti-p65 and anti-phospho- p65 were obtained from Bio-Rad (Oxford, UK).
3
Antibodies and Drugs for Protein Analysis
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The antibodies and drugs used in this study included rabbit monoclonal anti-Oct-1 antibody (Abcam, ab178869), rabbit monoclonal anti-HCF-1 antibody (Abcam, ab289975), mouse monoclonal anti-VP16 antibody (Santa Cruz, sc7545), mouse monoclonal anti-β-actin antibody (Sino Biological, 1000166), rabbit monoclonal anti-GAPDH antibody (Abways Technology, AB0037), mouse monoclonal anti-Histone-H3 antibody (Sino Biological, 100005-MM01), rabbit polyclonal antibody anti-TSG101 (Proteintech, 28283-I-AP), mouse monoclonal anti-HSV-1-gD antibody (Santa Cruz, sc21719), mouse monoclonal anti-ICP0 antibody (Santa Cruz, 13,118), Alexa Fluor 594-labeled goat anti-rabbit IgG (H + L; Invitrogen, 2165334), Alexa Fluor plus 488-labeled goat anti-mouse IgG (H + L; Invitrogen, A32723), goat anti-mouse IgG-HRP (Invitrogen, 31430), goat anti-rabbit IgG (H + L)-HRP (Invitrogen, 32460), and protease inhibitor cocktail (Thermo Scientific, EO0492).
4
Western Blot Analysis of Viral Proteins
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In brief, lysates were separated on polyacrylamide gels, transferred onto PVDF membranes, and blotted with the indicated antibodies: mouse monoclonal anti-Flag antibody (Abways, #AB0008), mouse monoclonal anti-ICP0 antibody (Santa Cruz, #sc-53070), mouse monoclonal anti-ICP8 antibody (Abcam, #ab20194), mouse monoclonal anti-VP16 antibody (Santa Cruz, #sc-7545), mouse monoclonal anti-β-actin antibody (Sino Biological, #1000166), anti-TK antibody (laboratory stock), rabbit anti-E-cadherin antibody (Affinity Biosciences, #AF0131), mouse monoclonal anti-GAPDH antibody (Abways, #AB0037), mouse monoclonal anti-Histone antibody (Sino Biological, #100005), rabbit polyclonal anti-dCas9 antibody (ABclonal, #A14997), goat anti-mouse IgG-HRP (Invitrogen, #31430), and goat anti-rabbit IgG (H+L)-HRP (Invitrogen, #32460).
5
Quantification of hACE2 Protein Expression
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The protein lysates were prepared from HEK-293T and 293T-hACE2 cells using RIPA buffer (Thermos scientific, VH310061) with protease inhibitor (Medchem Express, HY-K0010) and estimated protein concentration of each sample by the Bradford assay using BSA standards. 20 μg of protein lysates were resolved in 10% SDS-PAGE gel and transferred onto a PVDF membrane (WH3135834). After blocking with 5% non-fat dry milk powder in TBST, the blot was incubated with hACE2 antibody (Thermo Scientific, MA5-32307, and dilution at 1 in 2000 in blocking buffer) and followed by goat anti-rabbit IgG H&L-HRP (Invitrogen, ab205718, dilution of 1 in 10,000 in blocking buffer). The blot was visualized for the hACE2 band using a chemiluminescence substrate in Chem-Doc imaging system (Bio-Rad). For internal control, the blot was further stripped and probed for GAPDH (Biorad, MCA4740, dilution at 1 in 2000 in blocking buffer) with anti-mouse IgG antibody H&L-HRP (VectorLab, PI-2000, dilution at 1 in 10,000 in blocking buffer), and visualization of bands in the blot was performed.
6
Western Blot Analysis of TET Enzymes
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For detection of TET1 and TET2, mESCs cultured in standard mESCs medium were harvested after MEF depletion. For TET3, 8-10 day EBs were collected after differentiation in mESCs medium (supplemented with 20% serum, without 2i and LiF) in a petri dish. All the pellets were lysis in lysis buffer (150 mM sodium chloride, 1% triton x-100, 50 mM Tris HCl pH8.0 with freshly added proteinase inhibitor) for 30 min on ice. The supernatant was reserved for total protein quantification using Pierce BCA protein assay kit (Thermo Fisher Scientific, 23227). A total of 10-20 μg protein were loaded for SDS-PAGE analysis. Primary antibodies used in the assay: anti-Tet1 (Abcam, ab191698), anti-Tet2 (Abcam, ab124297), anti-Tet3 (GeneTex, GTX121453), anti-GAPDH (Abcam, ab181602). anti-alpha Tubulin (Millipore, ABT170). Secondary antibodies used: Goat anti-Rabbit IgG (H+L), HRP (Invitrogen, 31460), and Goat anti-Mouse IgG (H+L), HRP (Invitrogen, 31430). Signal was developed with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo, 34580), and images were taken using ChemiDoc™ MP Imaging System (BioRad).
7
Western Blot Analysis of Tat Mutants
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HEK293T cells transfected with WT and mutants Tat plasmids, and then treated with JQ1 (1 μM) or DMSO diluent control, were lysed with RIPA buffer (50 mM Tris–HCl pH8, 150 mM NaCl, 1% IgePal, 1 mM EDTA) supplemented with protease inhibitor cocktail (Roche), followed by sonication and centrifugation at 12,000xg for 15 min at 4 °C. The amount of proteins in the cell lysate was determined by Bradford assay (BioRad). Equal amounts of each sample were loaded on 12.5% SDS-PAGE, transferred to a nitrocellulose membrane (0.45 μm BioRad) then blocked in 5% milk-PBS-T (0.1% Tween-20) for 1 h at room temperature. Blots were probed with anti-Flag (ab1162, abcam, 1/2500°), anti-GAPDH (#14C10, cell signalling, 1/1000°), anti-PTBP1 (clone 7, ThermoFischer Scientific, #325000, 1/500°) and anti-hnRNP A1 (clone 9H10, Santa Cruz, sc-56700, 1/25°). After several washes, the membrane was incubated with either 1/5000° goat anti-rabbit IgG (H + L) HRP (Invitrogen, Cat. No. 656120) or goat anti-mouse IgG (H + L) HRP (Invitrogen, Cat. No. 626520) for 1 h at RT. Blots were developed using Supersignal west pico chemiluminescent substrate (ThermoFisher Scientific) and visualized using the MF-ChemiBis 3.2 imaging system (DNR).
8
ChIP-Seq and Western Blot Antibodies
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The following antibodies were used for ChIP: α-macroH2A1.2 (Millipore MABE61), α-phospho-S139-H2AX (Millipore 05–636), α-H2B (Abcam ab52484), α-H2A (Abcam ab18255), TRF2 (Novus Biologicals IMG-124A) and normal mouse IgG (Millipore 12–371). Primary antibodies for western blotting were: α-macroH2A1.2 (Millipore MABE61), α-phospho-S139-H2AX (S139) (Millipore 05–636), α-BRCA1 (Santa Cruz sc-6954), α-ATRX (Cell signaling 14820s), α-GAPDH (Santa Cruz sc-32233), α-H2AX (Abcam ab20669). Secondary antibodies were goat anti-mouse IgG (H+L)-HRP (Invitrogen 31430) and goat anti-rabbit IgG (H+L)-HRP (Invitrogen 31460). Primary antibodies for IF were α-BRCA1 (Santa Cruz sc-6954), α-γ-H2AX (Millipore 05–636, Abcam ab11174), α-BrdU (BD Biosciences 555627), and TRF2 (Novus Biologicals IMG-124A), secondary antibodies were goat-anti-mouse or goat-anti-rabbit IgG (H+L) coupled to Alexa Fluor 488 or 647 (Life Technologies).
9
Screening CRISPR Knockout Clones via Dot Blot
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To screen for potential knockout clones after CRISPR/Cas9-mediated gene editing, genomic DNA was extracted from each single clone after 5-6 passages post-transfection. A total of 50-100 ng genomic DNA was blotted onto nitrocellulose membranes with Bio-Dot microfiltration apparatus. Afterward, the membrane was air-dried and cross-linked on each side with a UV-agarose gel box. 5% non-fat milk in PBST was used to block the membrane, followed by primary and secondary antibody incubation. Antibodies used for this assay: anti-5hmC (ActiveMotif, 39069; Epigentek, A-1018), Goat anti-Rabbit IgG (H+L), HRP (Invitrogen, 31460), Goat anti-Mouse IgG (H+L), HRP (Invitrogen, 31430). Signal was developed with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo, 34579), and images were taken using ChemiDoc™ MP Imaging System (BioRad).
10
ChIP-Seq and Western Blot Antibodies
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