The following antibodies were purchased from Santa Cruz: monoclonal anti-HA, rabbit polyclonal anti-HA and polyclonal anti-Cdc20. Rabbit polyclonal antibodies to Histone H3, Cdc27 and PHistone-H2AX (Ser139) antibodies were purchased from Cell Signalling Technologies. The following antibodies were purchased from Sigma-Aldrich: monoclonal anti-g-tubulin, polyclonal α-tubulin, HRP-conjugated goat anti-mouse, HRP-conjugated goat anti-rabbit. A human anti-centromere antibody was purchased from Europa Bioproducts Ltd. Monoclonal antibodies to PCNA, Mad2B and c-myc were purchased from BD Transduction Laboratories. The following antibodies were purchased from Invitrogen: Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 594 rabbit anti-mouse IgG and Alexa Fluor 594 goat anti-human IgG.
Hrp conjugated goat anti rabbit
Manufactured by Merck Group
Sourced in United States
HRP-conjugated goat anti-rabbit is a secondary antibody that binds to rabbit primary antibodies. It is conjugated with horseradish peroxidase (HRP), an enzyme that can be used for detection in various immunoassays and immunohistochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated goat anti mouse, by Merck Group (6 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Rabbit polyclonal anti ha, by Santa Cruz Biotechnology (2 mentions)
Alexa fluor 594 rabbit anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 goat anti human igg, by Thermo Fisher Scientific (2 mentions)
Polyclonal anti cdc20, by Santa Cruz Biotechnology (2 mentions)
Polyclonal α tubulin, by Merck Group (2 mentions)
C myc, by BD (2 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Mouse anti flag, by Merck Group (2 mentions)
Phistone h2ax ser139, by Cell Signaling Technology (1 mentions)
Protease phosphatase inhibitor cocktail, by Roche (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence kit, by Merck Group (1 mentions)
Bca assay system, by Bio-Rad (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Super signal pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated goat anti mouse, by Santa Cruz Biotechnology (1 mentions)
14 protocols using hrp conjugated goat anti rabbit
1
Identification of Mitotic Regulators
2
ADAR1 Protein Expression in CRC Cells
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After rinsing with pre-cooled phosphate buffered saline, total protein in CRC (HCT-116 and HT29) cells was extracted with RIPA buffer (Thermo Fisher Scientific) and a protease/phosphatase inhibitor cocktail (Roche). Protein concentration was evaluated with a Bio-Rad BCA assay system, and equal amounts of protein were loaded into each lane of a polyacrylamide gel. Proteins were separated via SDS-PAGE, transferred to polyvinylidene difluoride membranes (Millipore, Billerica), and blocked with 5% skim milk. Membranes were stained with primary antibodies [anti-adenosine deaminase acting on RNA 1 (ADAR1) 1:500, Proteintech; anti-GAPDH 1:1000, Proteintech] for 16 h, then rinsed with TBST (3 × 10 min), and stained with secondary antibody (HRP conjugated goat anti-rabbit 1:1000, Sigma) at room temperature for 60 min. An enhanced chemiluminescence kit (Merck Millipore) and Kodak film (Kodak) was used to detect the blots. ADAR1 protein abundance was normalized to GAPDH.
3
Western Blot Analysis of RhoC, RhoA, and MRTF-A
4
Antimycin A and Oligomycin A Induced Mitochondrial Stress
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HeLa cells plated in 6-well plates were treated with 2 µM antimycin A and 4 µM oligomycin A for 2 and 10 min. For immunoblotting, whole cell lysates were prepared with radio immunoprecipitation assay (RIPA) buffer and 100 µg of total protein was subjected to SDS-PAGE. PVDF membranes were probed with primary polyclonal rabbit anti-MICU1 (Cell Signaling; 1:1000; 12524 S) and HRP-conjugated goat-anti-rabbit (Sigma; 1:1000; 12–348) antibodies. For normalization, membranes were stripped and re-probed with primary antibody against β-actin (Sigma; 1:1000; A5316). Immunoreactive bands were visualized using Immobilon Western HRP Substrate (Thermo Fisher) and the chemiluminescence detection system ChemiDoc (Bio-Rad).
5
Comprehensive Antibody Utilization Protocols
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The antibodies used in this study included mouse anti-Flag (Sigma, 1:3000), rabbit anti-Flag (Sigma, 1:2000), rabbit anti-Myc (MBL, 1:3000), mouse anti-GFP (Abmart, 1:5000), mouse anti-GAPDH (Sungene Biotech, 1:5000), mouse anti-α-Tubulin (Sungene Biotech, 1:5000), rabbit-anti-Oct4 (Santa Cruz, 1:2000), rabbit anti-HA (MBL, 1:3000), HRP-conjugated goat anti-mouse (Sigma, 1:3000), HRP-conjugated goat anti-rabbit (Sigma, 1:3000), goat anti-mouse Alexa555 (Molecular Probes, 1:2000) and goat anti-mouse Alexa488 (Molecular Probes, 1:2000).
6
Western Blot Analysis of PhyR in Brucella
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B. abortus cells were grown in TSB up to logarithmic phase, and equal amounts of bacteria of each strain were harvested by centrifugation for 2 min at 10,000 x g, resuspended in Laemmli sample buffer and inactivated by heating at 100°C for 20 min. These samples were then separated on a 15% SDS-PAGE and transferred to a nitrocellulose filter (Millipore). RibH1, a lumazine synthase (LS) isoenzyme, was used as loading control as it exhibits constitutive expression in B. abortus [43 (link), 44 (link)]. Membranes were probed with primary polyclonal mouse antiserum anti-PhyR (1:5,000), or polyclonal rabbit antiserum anti-RibH1 (1:2,000) in PBS-Tween 0.05% and 1% milk, with gently agitation at 4°C for 16 h. Membranes were then incubated with secondary antibody HRP-conjugated goat anti-mouse (1:2,000) (Sigma A4416) or HRP-conjugated goat anti-rabbit (1:5,000) (Sigma A6154), with gently agitation at room temperature for 1 h. Blots were developed using Pierce ECL Plus Western Blotting Substrate (Thermo Scientific), following the manufacturer’s instructions. Signal intensity was measured using a Storm 840 Molecular Imager (GE Healthcare), and then quantified using the ImageQuant 5.2 program.
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B. abortus cells were grown in TSB up to logarithmic phase, and equal amounts of bacteria of each strain were harvested by centrifugation for 2 min at 10,000 x g, resuspended in Laemmli sample buffer and inactivated by heating at 100°C for 20 min. These samples were then separated on a 15% SDS-PAGE and transferred to a nitrocellulose filter (Millipore). RibH1, a lumazine synthase (LS) isoenzyme, was used as loading control as it exhibits constitutive expression in B. abortus [43 (link), 44 (link)]. Membranes were probed with primary polyclonal mouse antiserum anti-PhyR (1:5,000), or polyclonal rabbit antiserum anti-RibH1 (1:2,000) in PBS-Tween 0.05% and 1% milk, with gently agitation at 4°C for 16 h. Membranes were then incubated with secondary antibody HRP-conjugated goat anti-mouse (1:2,000) (Sigma A4416) or HRP-conjugated goat anti-rabbit (1:5,000) (Sigma A6154), with gently agitation at room temperature for 1 h. Blots were developed using Pierce ECL Plus Western Blotting Substrate (Thermo Scientific), following the manufacturer’s instructions. Signal intensity was measured using a Storm 840 Molecular Imager (GE Healthcare), and then quantified using the ImageQuant 5.2 program.
7
Comprehensive Antibody Acquisition Protocol
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The following antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA): monoclonal anti-HA, rabbit polyclonal anti-HA and polyclonal anti-Cdc20. Rabbit polyclonal antibodies to Chk1, PChk1 (Ser317), Cdc27, PHistone-H2AX (Ser139), Histone H3 antibodies and mouse monoclonal FLAG antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). The following antibodies were purchased from Sigma-Aldrich (St Louis, MO, USA): monoclonal anti-γ-tubulin, polyclonal α-tubulin, horseradish peroxidase (HRP)-conjugated goat anti-mouse, HRP-conjugated goat anti-rabbit. A human anti-centromere antibody was purchased from Europa Bioproducts Ltd (Cambridge, UK). Monoclonal antibodies to Mad2B, Mad2A, PCNA and c-myc were purchased from BD Transduction Laboratories (Heidelberg, Germany). The following antibodies were purchased from Invitrogen (Carlsbad, CA, USA): Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 594 rabbit anti-mouse IgG and Alexa Fluor 594 goat anti-human IgG.
8
Quantitative Western Blot Assay for Serum Proteins
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Western blot analysis was performed to ensure the reliability of the HPLC-chip/MS results. The protein concentrations were determined by RCDC protein assay (Bio-Rad, USA). 30 μg or 10 μL serum was separated by 8% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Rabbit anti-alpha-1-antitrypsin (SERPINA1, Proteintech Group Inc., USA) and anti-alpha-2-antiplasmin (SERPINF2, Proteintech Group Inc., USA) were primary antibodies for the immunodetection. HRP-conjugated goat-anti-rabbit was used as secondary antibody (Sigma Aldrich, USA). Bands were visualized by chemiluminescence with ChemiDoc XRC+ Imaging System (Bio-Rad, USA) using ECL detection reagents (GE Healthcare, USA). Quantification of the detected bands density value was performed using the Quantity One software (version 4.6.2, Bio-Rad, USA). All immunoblots were run at least in triplicate.
9
Immunohistochemistry for Tyrosine Hydroxylase
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Free floating sections were incubated in 4% BSA for 1 h at RT and then incubated O/N at 4 °C with rabbit polyclonal anti-TH (1:500). In control sections, primary antibody was omitted. For blocking internal peroxidases 0.3% hydrogen peroxide solution for 10 min was used. After washes sections were incubated for 2 h at RT with HRP-conjugated goat anti-rabbit (1:100, Sigma, USA) in PBS 0.4% Triton X100. 3,3′-diamino-benzidine (DAB Substrate Kit for Peroxidase, Vector) was used as the chromogen. Sections were washed, and mounted with Eukitt® Quick-hardening mounting medium (Sigma, USA) and observed with a Leica S5. To quantify TH + cells, 3 slices were used per each group (n = 3).
10
Profiling Extracellular Vesicle Proteome
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EVs isolated from patients and controls were lysed in RIPA buffer containing complete mini protease inhibitors (Sigma) at 4°C for 30 minutes. Samples were sonicated for 2 seconds (three times), and spun at 13,000 × g for 30 minutes at 4°C. Protein concentrations were quantified by the BCA assay (Thermo Fisher Scientific). Protein samples were processed for immunoblotting and mass spectrometry (MS).
EV-derived proteins (20 µg) were separated using 12% Mini-PROTEAN® precast polyacrylamide gel (Bio-Rad). Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad). Membranes were blocked for 1 hour at room temperature with 5% nonfat dry milk in 1X Tris-buffer saline with 0.05% Tween 20 (TBST). Membranes were probed with anti-TSG101 (Abcam; 1:1,000) and anti-CD63 (Abcam; 1:1,000), anti-Alix (ThermoFisher Scientific 1:1,000), anti-β-actin (Sigma 1:1,000), anti-tenascin C (abcam 1:1,000), anti-vimentin (abcam 1:500) primary antibodies, followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Sigma 1:1,000) and goat anti-mouse (Sigma 1:3,000) secondary antibodies. Membranes were washed five times for 10 minutes each time after each incubation and developed using ECL prime Western blot detection (GE Healthcare). Protein signals were visualized using the ChemiDoc XRS + System.
EV-derived proteins (20 µg) were separated using 12% Mini-PROTEAN® precast polyacrylamide gel (Bio-Rad). Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad). Membranes were blocked for 1 hour at room temperature with 5% nonfat dry milk in 1X Tris-buffer saline with 0.05% Tween 20 (TBST). Membranes were probed with anti-TSG101 (Abcam; 1:1,000) and anti-CD63 (Abcam; 1:1,000), anti-Alix (ThermoFisher Scientific 1:1,000), anti-β-actin (Sigma 1:1,000), anti-tenascin C (abcam 1:1,000), anti-vimentin (abcam 1:500) primary antibodies, followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Sigma 1:1,000) and goat anti-mouse (Sigma 1:3,000) secondary antibodies. Membranes were washed five times for 10 minutes each time after each incubation and developed using ECL prime Western blot detection (GE Healthcare). Protein signals were visualized using the ChemiDoc XRS + System.
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