Thirty frozen NSCLC, 10 frozen SCLC, and 9 frozen normal lung tissues were lysed with 250 µL of RadioImmunoPrecipitation Assay (RIPA) buffer with anti-protease and anti-phosphatase agents (Roche Diagnostics, Mannhein, Germany). Equal amounts of lysate were separated by 12% SDS-PAGE and electrotransferred onto a nitrocellulose membrane. After blocking 1 h with 5% W/V BSA (Sigma, Saint-Louis, MO, USA) in TBS with 0.1% Tween 20 (Sigma), the membrane was incubated overnight at 4 °C with phospho-FAK Y397 rabbit antibody (1/1000 Cell Signaling Technology, Danvers, MA, USA) or total FAK mouse antibody (1/250, Santa Cruz Biotechnology, Dallas, TX, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rabbit antibody (1/5000, Sigma). Secondary antibodies consisted of HRP-conjugated goat anti-rabbit IgG (Cell Signaling Technology) or HRP-conjugated goat anti-mouse IgG (Sigma). Immunoreactivity bands were developed using chemiluminescence (Amersham ECL, GE Healthcare, Little Chalfont, Buckinghamshire, UK) and detected with a chemidoc XRS apparatus (Bio-rad, Hercule, CA, USA) and quantified using the Quantity One software (Bio-rad).
Hrp conjugated goat anti mouse igg
Manufactured by Merck Group
Sourced in United States, Germany, China, Spain
HRP-conjugated goat anti-mouse IgG is a secondary antibody used in immunological assays and research applications. It is composed of goat-derived polyclonal antibodies that are conjugated to the enzyme horseradish peroxidase (HRP). This conjugate can be used to detect and quantify mouse immunoglobulin G (IgG) in various experimental techniques.
Automatically generated - may contain errors
Lab products found in correlation
Immunospot microanalyzer, by Cellular Technology (20 mentions)
Hrp conjugated goat anti rabbit igg, by Merck Group (12 mentions)
Pvdf membrane, by Merck Group (8 mentions)
Trueblue peroxidase substrate, by Cellular Technology (6 mentions)
Horseradish peroxidase, by Merck Group (4 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (4 mentions)
Nitrocellulose membrane, by Merck Group (4 mentions)
Epoch microplate spectrophotometer, by Agilent Technologies (3 mentions)
Imagequant las 4000, by GE Healthcare (3 mentions)
3 3 diaminobenzidine (dab), by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Goat anti rabbit igg, by Merck Group (3 mentions)
Immunospot 5.0.37 macro analyzer, by Cellular Technology (2 mentions)
Tetramethylbenzidine tmb, by Merck Group (2 mentions)
Immunospot analyzer instrument, by Cellular Technology (2 mentions)
Ecl plus western blotting detection system, by GE Healthcare (2 mentions)
True blue substrate, by Merck Group (2 mentions)
Mouse anti hpiv3, by Abcam (2 mentions)
Rabbit anti β actin, by Proteintech (2 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit igg, by Merck Group (2 mentions)
151 protocols using hrp conjugated goat anti mouse igg
1
Phospho-FAK Y397 Expression in NSCLC and SCLC
2
Bacterial Protein Expression and Detection
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B.S. and B.S.-HA were grown in LB medium with 5 μg/ml chloramphenicol, adding IPTG (0.1 M) at growth phase (OD600 = 0.5), then grown for 3 h. The bacterial were washed three times with sterile phosphate-buffered saline (PBS) and collected. Then the B.S. and B.S.-HA was ultrasonicated. For immunodetection of the fusion proteins by western blotting13 (link), mouse anti-HA (Abcam, USA), followed by HRP-conjugated goat anti-mouse IgG (Sigma, USA) were used. Then the blots were developed by enhanced chemiluminescence.
3
Rat Corneal Protein Expression Analysis
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Rat corneal proteins were extracted with cold RIPA buffer. Aliquots having equal protein content were subjected to electrophoresis on 9% Tricine gels and then electrophorectially transferred to PVDF membranes. After 1 h blocking in 5% BSA, the blots were incubated with primary antibodies for: MK2(1:1000, Cell Signaling Technology, Danvers, MA, USA), Phospho-MK2(p-MK2, 1:1000, Cell Signaling Technology), p38(1:200, Santa Cruz Biotechnology Biotechnology, Dallas, TX, USA), Phospho-p38(p-p38, 1:1000, Cell Signaling Technology), PEDF(1:500, Abcam), VEGF(1:1000, Abcam) and HRP-conjugated anti-β-actin antibody (1:20,000, Sigma, Saint Louis, MO, USA) as a loading control. After washing each membrane three times with Tris-buffered saline containing 0.05% Tween 20 for 10 min, they were incubated with HRP-conjugated goat anti-rabbit IgG (1:10,000, Sigma) or HRP-conjugated goat anti-mouse IgG (1:10,000, Sigma) for 1 h at room temperature. The specific bands were visualized by an enhanced chemiluminescence reagent (ECL, Lulong Inc, Xiamen, China), and the image intensity was calculated with a transilluminator (ChemiDoc XRS System; Bio-Rad, Philadelphia, PA, USA).
4
Protein Expression and Localization Analysis
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Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting were used to analyze the expression of the target protein, and western blotting was developed using the gp85-specific mouse monoclonal antibody JE9 as the primary antibody. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Sigma, 1:5,000) was used as the secondary antibody.
For flow cytometry, L. plantarum cells were cultured in MRS broth overnight at 37°C. The cell pellets were sequentially incubated with gp85-specific mouse monoclonal antibody (1:800) and fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG secondary antibodies (1:5,000; Sigma). Finally, 3 × 104 cells were analyzed with a FACS Calibur platform (Becton Dickinson, Oxnard, CA, USA) equipped with CellQuest software.
For flow cytometry, L. plantarum cells were cultured in MRS broth overnight at 37°C. The cell pellets were sequentially incubated with gp85-specific mouse monoclonal antibody (1:800) and fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG secondary antibodies (1:5,000; Sigma). Finally, 3 × 104 cells were analyzed with a FACS Calibur platform (Becton Dickinson, Oxnard, CA, USA) equipped with CellQuest software.
5
Immunodetection of TGEV-N, p-EGFR, APN, and TLR-6
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For immunodetection of the TGEV-N, p-EGFR, APN and TLR-6 proteins by Western blotting [20 (link)], rabbit anti-TGEV (VMRD, Hangzhou, China), rabbit anti-p-EGFR (CST) and rabbit anti-TLR6 (Bioss), followed by HRP–conjugated goat anti-mouse IgG and HRP–conjugated goat anti-rabbit IgG (Sigma) were used. The signal was detected using Super Signal West Pico lit (Thermo Scientific) and subjected to Image Reader LAS-4000 imaging system (FUJIFILM, Japan). The intensity of the bands in terms of density was measured and normalized against GAPDH expression. Three independent experiments and appropriate gel exposures yielded very similar results for each treatment modality.
6
Quantifying Zika Virus Neutralization
7
SARS-CoV-2 Virus Neutralization Assay
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Serial dilutions of serum samples were incubated with 102 focus-forming units (FFU) of different strains of SARS-CoV-2 for 1 h at 37°C. Antibody-virus complexes were added to Vero-hTMPRSS2 cell monolayers in 96-well plates and incubated at 37°C for 1 h. Subsequently, cells were overlaid with 1% (w/v) methylcellulose in Eagle’s Minimal Essential medium (MEM, Thermo Fisher Scientific). Plates were harvested 30 h later by removing overlays and fixed with 4% paraformaldehyde (PFA) in PBS for 20 min at room temperature. Plates were washed and sequentially incubated with an oligoclonal pool of SARS2-2, SARS2-11, SARS2-16, SARS2-31, SARS2-38, SARS2-57, and SARS2-71 43 (link) anti-S protein antibodies and HRP-conjugated goat anti-mouse IgG (Sigma Cat # A8924) in PBS supplemented with 0.1% saponin and 0.1% bovine serum albumin. SARS-CoV-2-infected cell foci were visualized using TrueBlue peroxidase substrate (KPL) and quantitated on an ImmunoSpot microanalyzer (Cellular Technologies).
8
HA-Tagged Receptor Expression Quantification
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Cells were seeded at 125 000 per well
in a 48-well cell culture plate and allowed to adhere overnight. Plates
were washed with Tris-buffered saline (TBS; 50 mM Tris-HCl, 150 mM
NaCl, pH 7.5), fixed with 4% w/v paraformaldehyde for 30 min at RT,
and incubated with blocking buffer (1% w/v skim milk powder in 100
mM NaHCO3, pH 8.6) for 4 h at RT. Surface Human influenza
hemagglutinin (HA)-tagged receptors were detected using the HA-7 mouse
anti-HA antibody (1:1000, Sigma-Aldrich, Sydney, Australia), followed
by HRP-conjugated goat antimouse IgG (1:2000, Sigma-Aldrich). After
washing with TBS, the peroxidase substrate SIGMAFAST OPD was added,
and the reaction was terminated by the addition of 1 M HCl. The colored
reaction product was detected at 490 nm in a multilabel plate reader
(EnVision, PerkinElmer). The absorbance values for stably expressing
cells were normalized to those of untransfected cells.
in a 48-well cell culture plate and allowed to adhere overnight. Plates
were washed with Tris-buffered saline (TBS; 50 mM Tris-HCl, 150 mM
NaCl, pH 7.5), fixed with 4% w/v paraformaldehyde for 30 min at RT,
and incubated with blocking buffer (1% w/v skim milk powder in 100
mM NaHCO3, pH 8.6) for 4 h at RT. Surface Human influenza
hemagglutinin (HA)-tagged receptors were detected using the HA-7 mouse
anti-HA antibody (1:1000, Sigma-Aldrich, Sydney, Australia), followed
by HRP-conjugated goat antimouse IgG (1:2000, Sigma-Aldrich). After
washing with TBS, the peroxidase substrate SIGMAFAST OPD was added,
and the reaction was terminated by the addition of 1 M HCl. The colored
reaction product was detected at 490 nm in a multilabel plate reader
(EnVision, PerkinElmer). The absorbance values for stably expressing
cells were normalized to those of untransfected cells.
9
SARS-CoV-2 Neutralization Assay with Monoclonal Antibodies
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Serial dilutions of mAbs or sera were incubated with 102 focus-forming units (FFU) of different strains or variants of SARS-CoV-2 for 1 h at 37°C. Antibody-virus complexes were added to Vero-TMPRSS2 cell monolayers in 96-well plates and incubated at 37°C for 1 h. Subsequently, cells were overlaid with 1% (w/v) methylcellulose in MEM supplemented with 2% FBS. Plates were harvested 24 h later by removing overlays and fixed with 4% PFA in PBS for 20 min at room temperature. Plates were washed and sequentially incubated with an oligoclonal pool of SARS2–2, SARS2–11, SARS2–16, SARS2–31, SARS2–38, SARS2–57, and SARS2–714 (link)
73 (link). The anti-S antibodies and HRP-conjugated goat anti-mouse IgG (Sigma, 12–349) in PBS supplemented with 0.1% saponin and 0.1% bovine serum albumin. SARS-CoV-2-infected cell foci were visualized using TrueBlue peroxidase substrate (KPL) and quantitated on an ImmunoSpot microanalyzer (Cellular Technologies).
73 (link). The anti-S antibodies and HRP-conjugated goat anti-mouse IgG (Sigma, 12–349) in PBS supplemented with 0.1% saponin and 0.1% bovine serum albumin. SARS-CoV-2-infected cell foci were visualized using TrueBlue peroxidase substrate (KPL) and quantitated on an ImmunoSpot microanalyzer (Cellular Technologies).
10
Indirect ELISA for Chicken Immune Proteins
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Indirect ELISA was carried out using 4 different E. coli-expressed recombinant proteins (Lee et al., 2020b ), chIFN-γ (Yun et al., 2000 (link)), 3-1E (Lillehoj et al., 2005 (link)), chIL-10 (Lee et al., 2018b (link)), and chTNF-α (Lu et al., 2022a ). Briefly, flat-bottomed 96-well microtiter plates were coated with 1 µg/mL of each recombinant protein (3-1E, chIFN-γ, ChTNF-α, and chIL-10) overnight (O/N ) at 4°C. The plate was washed, blocked with Superblock Blocking Buffer (Thermo Scientific, Waltham, MA) at R/T for 1 h, and incubated with 1 µg/mL of mouse anti-3-1E mAbs (#312, #317, #318 #319, #320, and #323). Following washes, the plate was incubated with HRP-conjugated goat anti-mouse IgG (Sigma-Aldrich) in PBS (1:5,000) for 1 h at 37°C. The antigen-antibody reaction was visualized by same volume of 3,3′,5,5′-tetramethylbenzidine (TMB ) substrate (Sigma-Aldrich, St. Louis, MO) and the peroxidase reaction was stopped by 2 N H2SO4 (Sigma-Aldrich, St. Louis, MO). The absorbance (OD) was read at 450 nm by using an ELx-800 microplate reader (Biotek, Winooski, VT). All incubation steps were performed on the plate shaker, and the plates were washed 6 times with PBS-T after each step. Two independent experiments, each consisting of 3 replicates, were performed.
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