Urolithin A was purchased from Cayman Chemical Company (#22,607). Bafilomycin-A1 (#B1793) and anti-p62/SQSTM1 antibody (#P0067) were purchased from Sigma-Aldrich. Anti-amyloid beta peptide antibody (MOAB-2) was purchased from Millipore (#MABN254). AT8 phospho-Tau antibody (Ser202, Thr205) was purchased from Thermo Fisher Scientific (#MN1020). Anti-β-amyloid antibody (6E10) (#803,017) was purchased from Biolegend. LC3 antibody was purchased from Proteintech (#14,600–1-AP). Actin antibody was purchased from Cell Signaling Technology (#3700) goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor™ 488, was purchased from Sigma-Aldrich (#A-11001). DMEM/F12 (#11,330,032) and DQ-BSA red (#D12051) were purchased from Thermo Fisher Scientific. Earl’s balanced salt solution (EBSS) (#24,010–043) was purchased from Gibco. DMEM (#10–013-CV), fetal bovine serum (#35–010-CV) and penicillin–streptomycin solution (#30–002-CI) were purchased from Corning. Beta amyloid oligomers were generated according to the manufacturer’s instructions using the beta amyloid (1–42), aggregation kit (#A-1170–2) purchased from rPeptide.
Goat anti mouse igg h l
Manufactured by Merck Group
Sourced in United States
Goat anti-mouse IgG (H + L) is a laboratory reagent that recognizes both the heavy and light chains of mouse immunoglobulin G (IgG). It is commonly used in various immunological techniques, such as Western blotting, ELISA, and immunohistochemistry, to detect and quantify mouse IgG in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Anti p62 sqstm1 antibody, by Merck Group (1 mentions)
Anti β amyloid antibody 6e10, by BioLegend (1 mentions)
Dq red bsa, by Thermo Fisher Scientific (1 mentions)
Urolithin a, by Cayman Chemical (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Alexa fluor 488, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Corning (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
Penicillin streptomycin solution, by Corning (1 mentions)
Lc3 antibody, by Proteintech (1 mentions)
Anti mouse iga, by Merck Group (1 mentions)
Cell dissociation buffer enzyme free pbs based, by Thermo Fisher Scientific (1 mentions)
Lsrii sorp flow cytometer, by BD (1 mentions)
Trans blot semi dry transfer cell, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Gallios apparatus, by Beckman Coulter (1 mentions)
β actin, by Merck Group (1 mentions)
Ecl prime, by Cytiva (1 mentions)
P120ctn, by BD (1 mentions)
7 protocols using goat anti mouse igg h l
1
Amyloid β Oligomer Stimulation Protocol
2
Investigating Bcl-2, NF-κB, and GAPDH Levels
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Mouse anti Bcl-2 (cat. no., B9804; dilution, 1:1,000), NF-κB (cat. no., N8523; dilution, 1:1,000), GAPDH (cat. no., G9295; dilution, 1:35,000) were purchased from Sigma-Aldrich (Merck KGaA), the secondary antibody Goat Anti-Mouse IgG (H+L) (cat. no., SA00001-1; dilution, 1:8,000) was purchased from ProteinTech Group, Inc. Cultured A549 cells, A549/cis cells, A549/cis cells transfected with microRNA-152 mimics, and A549/cis cells transfected with unrelated microRNA-152 mimics were lysed in RIPA buffer with 1% phenylmethylsulfonyl fluoride; the latter two were cultured for 48 h after transfection. Total protein was determined using BCA method. Extracted proteins were loaded 50 µg and separated via 10% SDS-PAGE and electro-transferred onto a PVDF membrane. The blocking reagent was 5% skim milk, then the PVDF membrane was incubated overnight at 4°C in freshly prepared TBST. The blots were probed with primary antibodies at 4°C overnight and subsequently incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Signals were visualized using ECL substrates (Pierce; Thermo Fisher Scientific, Inc.). GAPDH was considered an endogenous control.
3
SDS-PAGE and Immunoblotting for IgG/IgA Detection
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SDS–PAGE and immunoblotting were performed under reducing and non-reducing conditions (Lu et al., 2013 (link)). In western blot assay, IgG/IgA was detected by the 1:2000 diluted HRP-conjugated goat anti-mouse IgG (H + L) and Anti-Mouse IgA (α-chain specific) − Peroxidase antibody produced in goat (Sigma, United States). The signals representing α-heavy and light chains were enzymatically detected using a chemiluminescence reagent (Pierce, United States).
4
Metabolic Labeling and Analysis of CHO Cells
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CHO
cells were treated at 20–25% confluency with Ac4GalNAz, Ac34FGalNAz, or DMSO in triplicate for 3 days
at which time cells were collected using cell dissociation buffer
enzyme-free PBS-based (Gibco) for 10 min at 37 °C and collected
by centrifugation (5 min, 1000g at 4 °C). Cells
were fixed in 4% paraformaldehyde in PBS on ice for 10 min and pelleted
(5 min, 1000g at 4 °C) before being washed twice
in ice-cold PBS. Primary antibodies (HS4C3 at 1:20; IO3H10 at 1:10;
GD3A12 at 1:10)21 (link),22 (link) were diluted in FACS buffer (0.2%
BSA in PBS) and incubated with cells for 1 h at 4 °C. Cells were
then washed two times with ice-cold PBS before incubation with anti-VSV
(P5D4) at 1:10 dilution in FACS buffer for 45 min at 4 °C and
then washed twice in ice-cold PBS. Goat anti-mouse IgG (H + L) and
Alexa Fluor 488 conjugate antibody (Sigma) were diluted 1:500 in FACS
buffer and incubated with cells for 45 min at 4 °C. Cells were
washed twice with ice-cold PBS before resuspension in 500 μL
of PBS for flow cytometry analysis. A total of 10,000 cells were analyzed
on a BD SORP LSRII flow cytometer using the 488 nm argon laser.
cells were treated at 20–25% confluency with Ac4GalNAz, Ac34FGalNAz, or DMSO in triplicate for 3 days
at which time cells were collected using cell dissociation buffer
enzyme-free PBS-based (Gibco) for 10 min at 37 °C and collected
by centrifugation (5 min, 1000g at 4 °C). Cells
were fixed in 4% paraformaldehyde in PBS on ice for 10 min and pelleted
(5 min, 1000g at 4 °C) before being washed twice
in ice-cold PBS. Primary antibodies (HS4C3 at 1:20; IO3H10 at 1:10;
GD3A12 at 1:10)21 (link),22 (link) were diluted in FACS buffer (0.2%
BSA in PBS) and incubated with cells for 1 h at 4 °C. Cells were
then washed two times with ice-cold PBS before incubation with anti-VSV
(P5D4) at 1:10 dilution in FACS buffer for 45 min at 4 °C and
then washed twice in ice-cold PBS. Goat anti-mouse IgG (H + L) and
Alexa Fluor 488 conjugate antibody (Sigma) were diluted 1:500 in FACS
buffer and incubated with cells for 45 min at 4 °C. Cells were
washed twice with ice-cold PBS before resuspension in 500 μL
of PBS for flow cytometry analysis. A total of 10,000 cells were analyzed
on a BD SORP LSRII flow cytometer using the 488 nm argon laser.
5
Immunoblot Analysis of Vibrio Virulence Proteins
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Proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and visualised by Coomassie blue staining. For immunoblots, proteins were electrophoretically transferred to a PVDF membrane (Millipore) using a Transblot semidry transfer cell (Bio-Rad). Membranes were blocked and incubated with antibodies in 5% dry milk, 20 mM Tris-500 mM NaCl, 0.05% Tween-20. The antibodies used were mouse polyclonal antisera against OmpU (dilution, 1:20,000) and IlpA (dilution, 1:10,000). The secondary antibody was goat-anti-mouse IgG (H?L) (Sigma). Proteins overexpressed in V. vulnificus such as Wza, BamD, Lsp, LptE, MFP, LolB, and LolE, which were tagged with a hexahistidine epitope tag, were detected directly with a nickel-activated HisProbe-HRP conjugate (Pierce). SuperSignal West Dura substrate (Pierce) was used for the chemiluminescent detection of HRP-labeled proteins.
6
Cell Surface Receptor Expression Analysis
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Cell surface receptor expression was analyzed in all cell lines with the following Abs: cetuximab (EGFR) (Merck), trastuzumab (HER2) (Roche), SGP1 (HER3) (Santa Cruz Biotechnology), and H4.77.16 (HER4) (Thermo Fisher Scientific). Cetuximab and trastuzumab were purchased from the pharmacy of the ICM hospital. Exponentially growing cells were harvested and resuspended in FACS buffer (PBS/1% FBS). Cells (3 × 105) were incubated with 10 μg/mL primary Ab on ice for 1 hour 30 minutes. For competition experiments, cells were incubated with 15 μg/mL of the selected anti‐HER4 mAbs and NRG1 at various concentrations (0.1‐100 nmol/L; 3‐3000 ng/mL) on ice for 1 hour 30 minutes. After washes, cells were incubated with FITC‐conjugated goat anti‐human IgG (Fc specific) (Sigma) or goat anti‐mouse IgG (H + L) (Millipore). The cell median fluorescence intensity was measured by flow cytometry (Gallios apparatus; Beckman Coulter).
7
Western Blot Analysis of p120ctn
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Cell protein lysates were harvested by rinsing plates in PBS and using a lysis buffer consisting of Tris-HCL, NaCL, NP-40 and phosphatase and protease inhibitors as described previously [9 (link)]. Western blotting was performed as described previously [21 (link)]. Briefly, a 10% denaturing acrylamide gel was used to separate 10ug of the protein lysates and then transferred to a PVDF membrane. Primary antibodies, p120ctn (BD Transduction Labs #610134 – 1:10,000 dilution) and b-actin (Sigma-Aldrich #A5316 – 1:10,000 dilution), were incubated overnight at 4°C. Secondary antibody, Goat anti-mouse IgG (H+L) (Millipore #AP124P – 1:5000 dilution), was incubated at room temperature for 1 hour. ECL Prime (Cytiva Amersham #RPN2232) was used to develop the western blot and was read on a FluorChem R System (ProteinSimple).
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