Brain samples from non-treated mice were homogenized (10% wt/vol) in ice-cold lysis buffer—50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% (wt/vol) Triton X-100, 0.5% (wt/vol) Nonidet P-40 (IGEPAL; Sigma), glycerol 10%, 1 mM EDTA, 1 mM EGTA, and protease and phosphatase inhibitors—using a motor-driven, glass-Teflon homogenizer in ice, and then centrifuged at 15,000g for 20 min. Protein concentration was quantified with BCA kit (Pierce). Protein extracts were boiled in Laemmli sample buffer at 100 °C for 5 min. Equal amounts of total protein were separated with 6–10% SDS-PAGE electrophoresis, electrotransferred to polyvinylidene fluoride (PVDF) membranes (Millipore), and probed with indicated antibodies. Visualization of bound antibodies was performed using goat anti-mouse HRP (1:4000 diluted; Dako, Glostrup, Denmark) and the ECL Plus kit (Amersham-Pharmacia Biotech, GE Healthcare Bio-Sciences, Piscataway, NJ, USA).
Goat anti mouse hrp
Manufactured by Agilent Technologies
Sourced in Denmark, United States, Germany
Goat anti-mouse HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and bind to mouse primary antibodies, enabling visualization and quantification of target proteins in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit hrp, by Agilent Technologies (11 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
P0447, by Agilent Technologies (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
3 3 5 5 tetramethylbenzidine substrate, by Thermo Fisher Scientific (2 mentions)
Infinite f200, by Tecan (2 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (2 mentions)
Mouse anti np igg1 clone aa5h, by Bio-Rad (2 mentions)
Trans blot turbo system, by Bio-Rad (2 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Immobilon western, by Merck Group (2 mentions)
Cytation 3 imaging reader, by Agilent Technologies (2 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Swine anti rabbit hrp, by Agilent Technologies (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ecl plus kit, by Cytiva (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Streptavidin horseradish peroxidase hrp, by Agilent Technologies (1 mentions)
2 n h2so4, by Merck Group (1 mentions)
38 protocols using goat anti mouse hrp
1
Brain Protein Extraction and Immunoblotting
2
Fixed-cell ELISA for Virus Detection
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Fixed-cell ELISA was performed as per previously published methods with slight modifications [18 (link)]. Briefly, infected or mock-infected cell monolayers were fixed with acetone fixative buffer. Plates were blocked with 150 μl per well ELISA blocking buffer (0.05 M Tris/HCl (pH 8.0), 1 mM EDTA, 0.15 M NaCl, 0.05% (v/v) Tween 20, 0.2% w/v casein) for 1 hour at room temperature before probing with 50 μl/well respective mAbs diluted in blocking buffer as needed. Plates were incubated with primary antibody for 1 hour at 37°C before washing 4x with PBS containing 0.05% tween-20 (PBST). Secondary antibody goat anti-mouse HRP (DAKO) was diluted 1/2000 in blocking buffer and added at 50 μl/well. After another incubation at 37°C for 1 hour, plates were washed 6x with PBST. Finally, 100 μl/well substrate solution [1 mM 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 3 mM H2O2 in a buffer prepared by mixing 0.1 M citric acid with 0.2 M Na2HPO4 to give a pH of 4.2] was added per well and plates were incubated in the dark at room temperature for 1 hour. Absorbance was measured at 405 nm.
3
MERS-CoV Antibody Titer Quantification
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MERS-CoV–specific antibody titers were measured by ELISA. First, 96-well plates were coated with MERS-CoV S1 or MERS-CoV RBD proteins at 1 μg/ml in PBS (pH 7.4) and incubated overnight at 4°C. Wells were then washed three times with PBS, blocked with 10% normal goat serum in PBS, and incubated at 37°C for 30 min. Dromedary camel sera or VHHs were serially diluted in PBS, 100 μl was added per well, and plates were incubated at 37°C for 1 hour. Next, plates were washed three times in PBS containing 0.05% Tween 20 (PBST), after which they were incubated with biotin-conjugated goat anti-llama antibodies (1:2000, Abcore) or mouse anti-histidine antibodies (1:2000, Thermo Fisher Scientific) at 37°C for 1 hour. After three washes with PBST, plates were incubated with streptavidin horseradish peroxidase (HRP; 1:10,000, Dako) or goat anti-mouse HRP (1:2000, Dako) at 37°C for 1 hour. After this incubation, plates were washed three times in PBST and incubated at room temperature for 10 min in the presence of 3,3′,5,5′-tetramethylbenzidine substrate (eBioscience). Reactions were stopped with 2N H2SO4 (Sigma). The absorbance of each sample was read at 450 nm with an ELISA reader (Tecan Infinite F200).
4
Immunoblotting and Co-immunoprecipitation Protocols
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Immunoblotting was done overnight at 40 V onto PVDF membrane (BioRad). For immunostaining of co-immunoprecipitation samples from HEK293T cells the following antibodies were used: anti-V5 (Abcam, 1∶1,000), anti-HA (3F10, Roche, 1∶1,000) in 5% milk TBST, incubation was done overnight at 4°C on a shaking platform. For co-immunoprecipitation samples from brain anti-PSD95 antibody (75-028, Neuromab, 1∶10,000) was used. The secondary antibodies used were goat-anti-mouse-HRP (DAKO, for anti-V5 and anti-PSD95) and goat-anti-rat-AP (Southern Biotech, for anti-HA). The membranes were imaged by means of enhanced chemifluorescence (Amersham) or enhanced chemiluminescence femto (Thermo Scientific) according to the manufacturer's instructions.
5
EV Marker Detection by Western Blotting
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Isolated EVs were tested for the presence of the EV markers TSG101 and Flot1 using western blotting. The concentrated EV fractions without protein contamination were lysed in SDS sample buffer (4x Laemmli sample buffer, BioRad, CA, USA) with 1.4-Dithiothreitol (DTT) for Flot1 and without DTT for TSG101 (non-reducing), separated by SDS-PAGE (12% Criterium TGX Precast Gels, BioRad) and transferred to mid-size transfer stacks (Trans-Blot Turbo, BioRad). Strips of the blots were blocked in 2% BSA-PBS and incubated with primary antibodies against flotillin 1 (1:500, 610821, BD Biosciences, CA, USA) and tsg101 (1:500, Ab83, Abcam, UK) followed by the secondary antibody, goat-anti-mouse-HRP (Z0334, Dako, CA, USA). Chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, Thermo Scientific) was recorded using a PXi4Touch (Syngene, UK).
6
Specificity of Anti-MERS-CoV S1 Nanobodies
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The specificity of anti-S1A, anti-S1B, and control nanobodies for MERS-CoV S1, S1A, and S1B proteins was determined using ELISAs as previously described (29 (link)). In brief, 96-well ELISA plates were coated with 1 μg/ml MERS-CoV S1 (amino acids 1 to 751), S1A (amino acids 1 to 357), or S1B (amino acids 358 to 588) protein in phosphate-buffered saline (PBS) (pH 7.4) and incubated overnight at 4°C. Wells were then washed with PBS and blocked with 1% bovine serum albumin in PBS–0.5% Tween 20 for 1 h at 37°C. Nanobodies were 2-fold serially diluted in blocking buffer starting at a 1-μg/ml concentration, 100 μl of each dilution was added per well, and plates were incubated at 37°C for 1 h. Next, plates were washed three times in PBS–0.05% Tween 20 (PBST), after which they were incubated with mouse anti-His tag antibodies (1:2,000; Thermo Fisher Scientific) at 37°C for 1 h. Following incubation, the plates were washed and further incubated with goat anti-mouse HRP (1:2,000; Dako) at 37°C for 1 h. After this incubation, plates were washed three times in PBST, a 3,3′,5,5′-tetramethylbenzidine substrate (eBioscience) was added, and the plates were incubated for 10 min. The reaction was stopped with 0.5 N H2SO4 (Sigma). The absorbance of each sample was read at 450 nm with an ELISA reader (Tecan Infinite F200).
7
Immunohistochemical Analysis of Intestinal Cell Proliferation
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Intestinal sections were deparaffinized before blocking endogenous peroxidases by incubation in 1% H2O2 in methanol, and heat-induced antigen retrieval in 10 mM citric acid buffer (pH 6). BrdU- and IdU-labeled cells were detected with sheep anti-BrdU-biotin or mouse anti-IdU (AbCam, Cambridge, United Kingdom) followed by NeutrAvidin-horseradish peroxidase (HRP; Thermo Fisher Scientific, Loughborough, United Kingdom) or goat-anti-mouse-HRP (Dako, Glostrup, Denmark) detected using 3,3′-diaminobenzidine (DAB; Dako) and counterstained with hematoxylin. Mitoses were detected with rabbit monoclonal Ab (EPR17246) to histone 3 (H3) (phospho-S10; AbCam). Expression of omomyc in intestinal crypts and villi was detected, using rabbit anti-omomyc antibody, goat anti-rabbit-HRP secondary, and DAB detection, as above. Intestinal cryosections prepared at 5 μm were fixed in acetone/methanol before incubation with primary antibodies to BrdU (BU1/75-DyLight 550; Novus Biologicals, Abdingdon, United Kingdom) and/or against IdU (mouse anti-IdU clone 32D8.D9; AbCam) followed by goat-anti-mouse Alexa 488 (Thermo Fisher Scientific).
8
Antibody and Staining Protocol for Cellular Imaging
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Rabbit anti-vimentin antibody (ab16700), rabbit anti-tubulin (ab18251), mouse anti-LAMP1 (ab25630) were from (Abcam, Cambridge, UK). Mouse anti-tubulin (T5168, Sigma-Aldrich), rabbit anti-LC3 (0260–100 LC3-2G6, Nanotools, Teningen, Germany), rpS6 (2317) and phosphorylated rpS6 (p-rpS6 S235/236, 4856) were from (New England Biolabs, Herts, UK). Secondary antibodies for immunostaining: anti-rabbit FITC and anti-rabbit TRIC were from (Sigma-Aldrich), anti-rabbit Alexa Fluor 647 (A21246, ThermoFisher, Paisley, UK), anti-rabbit Alexa Fluor 488 (A11034, Invitrogen), anti-mouse Alexa Fluor 568 (A11004, Invitrogen,). Actin staining was performed using Alexa Fluor 594 Phalloidin (A12381, Invitrogen,) or Alexa Fluor 488 Phalloidin (A12379, Invitrogen,). LysoTracker Red (L7528, ThermoFisher) was used to visualise lysosomes in live cell imaging. Secondary antibodies for western immunoblotting: goat anti-rabbit HRP (P0448, Dako, Santa Clara. USA) and goat anti-mouse HRP (P0447, Dako).
9
Monoclonal Antibody Neutralization Assay
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A microneutralization (MN) assay was used to determine the neutralising concentrations of the mAbs. Briefly, viruses were titrated to give a plateau expression of nucleoprotein (NP) in 96-well flat-bottomed plates. Viruses and mAbs were separately diluted in virus growth medium (VGM) containing DMEM, 100 IU/ml penicillin, 100 µg/mL streptomycin and 0.1% bovine serum albumin (BSA). mAbs were serially diluted 1:2 in 50µl. Fifty microliters of the diluted virus was added to 50 µL mAb and incubated for 1 h at 37°C. 100 µL of 3 × 104 MDCK-SIAT1-HA cells were added to the wells containing the virus and mAb mixtures and incubated overnight at 37°C. The media was removed, and the monolayer was fixed with 4% paraformaldehyde, permeabilized with Triton-X100 and stained with mouse anti-NP IgG1 (clone AA5H, Bio-Rad Antibodies) followed by goat anti-mouse HRP (DAKO) antibody. TMB substrate was added and incubated for 2-5 min and the reaction stopped with 50 uL of 1M sulphuric acid followed by absorbance measurement as above. The neutralizing concentration of mAbs was defined as the concentration that caused a 50% reduction in NP expression.
10
Western Blot Analysis of α-Synuclein in Aged Mouse Brain
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For western blotting, we isolated individual hemispheres, excluding the olfactory bulb and cerebellum, from 16-month-old mice. These hemispheres were then lysed in 1 ml of ice-cold RIPA buffer (comprising 50 mM Tris HCl, 150 mM NaCl, 1 mM MgCl2, 1.0% NP-40 (v/v), 0.5% sodium deoxycholate (w/v), 1 mM EDTA, 0.1% SDS (w/v), pH 7.4), and supplemented with phosphatase and protease inhibitors. The protein concentration was determined using a BCA kit (BCA Protein Assay-Kit, Thermofisher). Following this, proteins were separated by SDS-PAGE and subsequently transferred to nitrocellulose membranes (BioRad) using the TransBlot Turbo system. The membranes were blocked for 1 h using 3% skim milk (w/v) in TBS-T. They were then incubated overnight with mouse primary antibodies targeting human α-Syn LB509 (1:500, #ab27766, Abcam), α-Syn (1:500, #610787, BD), GAPDH (1:500, MAB374, Millipore), p-Ser129-α-Syn (1:500, #825701, BioLegend) or β-actin (1:40,000, #A5441, Sigma). After washing, the membranes were exposed to a secondary antibody, goat anti-mouse-HRP (1:10.000, #P0447, Dako) for 1 h and subsequently reacted by chemiluminescence using SuperSignal (Thermo Fisher Scientific). Chemiluminiscence was imaged by an Alliance MINI HD 6 analyzer (UVITEC, UK) and protein expression was calculated by quantifying western blot band intensities using the ImageJ Gel Analyzer plugin.
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