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9 protocols using hrp conjugated goat anti mouse igg

1

Urea-Mediated Antibody Dissociation Assay

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Immunized serums were diluted 1:1000 in PBS containing 1% BSA and added to 96-well plates coated with recombinant IsdB (1μg/ml). After incubation for 1 hr, different concentration of urea (0 to 8 M) with 0.05% Tween 20 in PBS were treated for 15 mins. Plates were washed and bound antibodies were detected by horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (BioLegend, 405306).
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2

Immunoassay for Amyloid-Beta Peptides

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Aβ40 or Aβ42 (Anaspec, USA) was coated on 96-well microplates overnight at 4°C, then blocked with blocking buffer (2% BSA+5% goat serum in PBS) for 2 h at room temperature. The serum samples diluted into 1:1000 were added to the plates and incubated 2 h at room temperature. After washing, HRP-conjugated goat anti-mouse IgG (Biolegend) was added to the plates and incubated for 1 h. The reaction was developed by TMB substrate (Thermo Scientific, USA) and stopped with 0.1 N HCl. The microplate was read at 450 nm under a microplate reader (Bio-Tek, ELX800, USA). The antibody concentrations were calculated using a standard curve generated with known concentrations of anti-Aβ antibody.
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3

Western Blot Protein Detection

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Cells were lysed with lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol, supplemented with a protease inhibitor cocktail (Halt™ Protease inhibitor cocktail, Thermo Scientific)). Proteins in the cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto nitrocellulose membranes (Bio-Rad Laboratories). The membrane was blocked in 5% skim milk prior to incubation with indicated primary antibodies. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Biolegend) or (HRP)-conjugated donkey anti-rabbit IgG (BioLegend) was used as secondary antibodies. Primary antibodies used in this study include mouse-anti-Myc (Thermo Scientific), rabbit-anti-FLAG (Cell Science Technology), rabbit-anti-HA (Cell Science Technology), mouse-anti-S1 (a kind gift from Dr. Qigai He, Huazhong Agricultural University), and mouse-anti-PEDV N (SD 6–29, Medgene Labs).
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4

Screening Hybridoma Supernatants for MHCII Specificity

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Twelve days post-HAT selection, individual colonies were handpicked and transferred to 96-well plates containing medium E (Stem cell technologies). Four days later hybridoma supernatants were transferred to 96-well plates and fresh medium E was added to the cells. To test the specificity of hybridoma supernatants for MHCII and p:MHCII, a decoy screening approach was employed. ELISA plates were coated with 50 ng well−1 either p63:IAg7 or InsB10–23:IAg7 (for p63:IAg7 antibodies), or 2W:IAb or LLO:IAb, and then blocked with 1% BSA in PBS for 1 h. Hybridoma supernatants were mixed 1:1 with ELISA wash buffer (PBS+0.05% Tween20) and added to the p:MHCII-coated plates and incubated at 37 °C for 2 h. Media alone was used as a negative control while anti-IAg7 (clone 10–2.16 (ref. 28 (link)), BioXcell) or anti-IAb (Y3P29 (link), ATCC) were used as a positive control. For antibody detection the wells were incubated with HRP-conjugated goat anti-mouse IgG (BioLegend) diluted to 1:2,000 at room temperature for 2 h followed by addition of ABTS substrate solution (KPL) and detection by absorbance at 405 nm. Antibodies reacting to both p:MHCII monomers were considered specific for MHCII independent of peptide, while antibodies reacting with only p63:IAg7 or 2W:IAb were considered p:MHCII specific.
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5

ELISA for Amyloid-β Quantification

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Aβ40 or Aβ42 (Anaspec, USA) was coated on 96-well microplates overnight at 4 ℃, then blocked with blocking buffer (2% BSA + 5% goat serum in PBS) for 2 h at room temperature. The serum samples diluted into 1:1000 were added to the plates and incubated 2 h at room temperature. After washing, HRP-conjugated goat anti-mouse IgG (Biolegend) was added to the plates and incubated for 1 h. The reaction was developed by TMB substrate (Thermo Scientific, USA) and stopped with 0.1 N HCl. The microplate was read at 450 nm under a microplate reader (Bio-Tek, ELX800, USA). The antibody concentrations were calculated using a standard curve generated with known concentrations of anti-Aβ antibody.
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6

Quantitative ELISA for Antibody Measurement

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Total and antigen-specific antibody levels in human and mouse sera were measured by ELISA. Sera were serially diluted in PBS containing 1% BSA and added to 96-well high-binding plates coated with recombinant proteins (10 μg/ml). Bound antibodies were detected by horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG or donkey anti-human IgG (BioLegend). Quantitative antibody measurements were performed using commercially purchased human and mouse IgG standards (ThermoFisher Scientific)
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7

Antibody Validation for PARP1 Detection

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Antibodies used for Western blots included: anti-rec-PARP1, a rabbit polyclonal raised against recombinant PARP1 (Enzo Life Sciences ALX-210–302-R100; 1:4000 dilution); anti-N-ter-PARP1, a rabbit polyclonal raised against the N-terminal half of recombinant PARP1 (Active Motif Ab 2793257, 1:2000 dilution); anti-C-ter-PARP1, an affinity-purified mouse mAb that recognizes an epitope in the C-terminal NAD binding and catalytic domain of human PARP1 (clone 7D3–6, BD Biosciences #556493; 1:500 dilution); anti-cc-PARP1, a mouse mAb specific for the neo-epitope generated at the N-terminal of the 89 kD PARP1 fragment formed after cleavage by apoptotic caspases between Asp214/Gly215 (clone F21–852, BD Biosciences #552596, 1:1000 dilution). Secondary detection was with HRP-conjugated goat anti-mouse IgG or donkey anti-rabbit IgG (BioLegend, #405306 and #406401, respectively; 1:5000 dilution). Immune complexes were visualized using the SuperSignal West system (Pierce). Specificity of PARP1 Abs for full-length 113 kD PARP1 and its cleavage products was assessed by Western blots of whole cell extracts treated with staurosporine (EMD Millipore; 1 μM, 1 hr), a kinase inhibitor that induces apoptotic PARP1 cleavage (Fig. S1A).
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8

SARS-CoV-2 Antibody Quantification ELISA

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Ninety-six-well plates were coated with 1 μg/ml SARS-CoV-2 S1 protein (Sino Biological Inc.) or S1 RBD protein (Sino Biological Inc.) in PBS and incubated at 4°C overnight. After coating, plates were treated with 2% bovine serum albumin (BSA) (Sigma)/PBS for 2-5 h at 4°C. After blocking, serially diluted heat-inactivated sera or nasal washes (1%BSA/PBS) from the vaccinated or control mice were added to wells, and the plates were incubated for 1.5 h at RT, followed by washing 6 times with PBST and subsequent 1h incubation with an HRP-conjugated goat anti-mouse IgG (1:3,000 dilution; BioLegend) or goat anti-mouse IgA antibody (1:5,000 dilution; SouthernBiotech) in 1% BSA/PBS at RT. The wells were washed 6 times with PBST, and 100 μL of the TMB substrate set (BioLegend) was added to the immune complexes in each well. Plate development was then halted by the addition of 50 μL of 2N H2SO4 per well. The absorbance at 450 nm was recorded using a microplate reader. The ELISA endpoint titers were defined as the highest reciprocal serum dilution that yielded an absorbance >0.2. (Log10 endpoint titers were given).
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9

Western Blot Analysis of Cell Signaling Proteins

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Protein samples were separated in polyacrylamide gels (7 to 12%, depending on the target protein) and transferred to nitrocellulose membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Western blotting (WB) was performed as described previously [56 (link)]. Primary antibodies used were anti-CD6 mAb (MEM98, EXBIO), anti-RasGAP mAb (B4F8, Santa Cruz Biotechnology), anti-p21 Ras mAb (Cytoskeleton, Inc), anti-α-tubulin mAb (B-5-1-2, Merck), anti-ERK 2 rabbit polyclonal (C14, Santa Cruz Biotechnology), and Phospho-p44/42 MAPK rabbit polyclonal (Cell Signaling Technology); secondary antibodies used were HRP-conjugated goat anti-mouse IgG (BioLegend), rat anti-mouse IgG (Abcam) for detection of CD6 in MEM98 immunoprecipitates, and mouse anti-rabbit IgG (Santa Cruz Biotechnology). Membranes were developed with Amersham ECL Select/Prime Western Blotting Detection Reagent (Cytiva). Data were acquired using the ChemiDoc XRS+ System (Bio-Rad) and analyzed by Fiji [57 (link)].
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