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4 protocols using horseradish peroxidase conjugated goat anti mouse igg secondary antibody

1

Calmodulin Binding Assay Using GST Fusion

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Purified GST fusion proteins immobilized on GSH-Sepharose 4B beads (GE Healthcare) were equilibrated in pull-down buffer (25 mM Hepes, 120 mM KCl, 5 mM NaCl, pH 7.5) with either 100 μM CaCl2 or 5 mM EGTA. Calmodulin (10 μg) was added to the beads and incubated for 45 min at room temperature. After three washes, the proteins were recovered, separated by 15% SDS-PAGE, and transferred to Immobilon-P PVDF membrane (Merck Millipore) for Western blotting. The membrane was blocked with 5% nonfat milk in TBS-T (10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.5), incubated with the primary antibody (monoclonal anti-CaM 05–173 from Merck Millipore, diluted 1:2000 in blocking buffer) O/N at 4°C, washed, and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (Bio-Rad) diluted 1:5000 in blocking buffer. Antibody binding was detected using enhanced chemiluminiscence and ECL hyperfilm (GE Healthcare). GST-fusion proteins (input) were identified using an Anti-GST–Peroxidase Conjugate antibody (dilution 1:5000; A7340 Sigma). ImageJ software (Version WCIF, National Institutes of Health, USA) was used to quantify band intensities and calculate the relative CaM binding % (n = 3).
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2

Quantitative Western Blot Analysis of TREM2

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Briefly, proteins were denatured in SDS loading buffer to a final concentration of 5 mg/ml. Proteins (50 μg/sample) were separated by 4–20% SDS-PAGE (Lonza, Rockland, ME) and electrophoretically transferred to polyvinylidene fluoride membranes (Immobilon P, Millipore) (Perez et al., 2015 (link))]. Membranes were blocked in Tris-buffered saline (TBS)/ 0.05% Tween-20/ 5% milk (1 h) at room temperature (RT). TREM2 or Iba1 antibodies were added to blocking buffer and membranes were incubated overnight (4o C), washed, incubated at RT (1 h) with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (1:8000, Bio-Rad, Hercules, CA) or goat-anti rabbit IgG secondary antibody (1:5000, Bio-Rad, Hercules, CA), visualized by chemiluminescence on a Kodak Image Station 440CF (Perkin-Elmer, Wellesley, MA) and quantified with Kodak 1. The ~ 25 kDa TREM2 band was quantified across clinical groups. Protein signals were normalized to β-tubulin and analyzed in three independent experiments (Perez et al., 2015 (link)). Immunoblot controls consisted of pre-adsorption of the TREM2 antibody with an excess of the immunogenic TREM2 peptide (Abcam, ab217864), that blocks the anti TREM2 antibody-N-terminal used in the study (Abcam, ab175262) and deletion of the primary, both resulting in the absence of immunoreactive bands in the membranes (see Fig. S1).
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3

Western Blot Analysis of hnRNP A1 in Neuro-2a Cells

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Neuro-2a cells were plated onto poly-D-lysine (Sigma-Aldrich) coated six-well plates and transfected as described above and harvested for Western blotting at 72 h after siRNA transfection. Neuro-2a cells were lysed in CytoBuster (Millipore) protease extraction reagent containing protease inhibitors (Roche) as per the manufacturer’s protocol. Cell lysates were separated by SDS-PAGE before being transferred to PVDF membrane. Membranes were blocked in 10% normal goat serum for 1 h at room temperature before being placed into primary antibody for overnight incubation at 4°C. The following primary antibodies were used: mouse anti-β-actin (1:1000; Cell Signaling Technology, RRID: AB_2242334) and mouse anti-hnRNP A1 (1:1000; Millipore, RRID: AB_10562650). Membranes were washed and incubated with goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (1:3000; Bio-Rad, RRID: AB_11125547). Membranes were developed using Clarity Western ECL substrate (Bio-Rad) and visualized using the Bio-Rad ChemiDoc system. Three biological replicates of siNEG and siA1 transfected Neuro-2a cells were harvested and run for Western blotting. Blots were analyzed in ImageJ (RRID: SCR_003070) by densitometry and normalized to β-actin signal.
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4

Quantifying Laminin γ2 Protein in Skin

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Skin samples were pulverized and homogenized with stainless steel grinding beads (OPS Diagnostics, LLC, Lebanon, NJ) using a GenoGrinder in the presence of protease inhibitors (Complete Mini, EDTA-free Protease Inhibitor Cocktail Tablets, Roche) in RIPA buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1% Nadeoxycholate, and 0.1% SDS) and assayed for protein concentration using BCA Protein Assay Reagent (Pierce Chemical, Rockford, IL). Pooled protein lysates (12 μg, N = 10 per group) were analyzed using 5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Life Sciences Group, Hercules, CA). The membranes were blocked with 5% nonfat milk solution and incubated with a mouse anti-laminin γ2 monoclonal antibody (1:100, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4°C, followed by goat anti-mouse IgG horseradish peroxidase conjugated secondary antibody (1: 6,000; BioRad). Proteins were detected by chemiluminescence (SuperSignal West Pico Chemiluminescence Substrate System, Pierce Chemical). GAPDH was used to monitor equal protein loading.
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