Whole cell extracts were prepared by resuspending cells in RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% NP-40 substitute, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with complete protease inhibitor cocktail (Roche). Cleared lysates were boiled in Laemmli sample buffer with 1% beta-mercaptoethanol and proteins were separated by SDS-PAGE using precast 4–12% Bis-Tris gels (Life Technologies). Proteins were transferred to PVDF membrane (Millipore) at 30 V, 4 °C for 16 h. Blots were blocked for 30 min with 5% milk in TBS-T, incubated with primary antibodies for >3h and horseradish peroxidase conjugated anti-mouse IgG or anti-rabbit IgG (GE Healthcare) for 1 h, washing 4× for 10 min with excess TBS-T in between and prior to developing. Blots were developed with SuperSignal West Pico ECL substrate (Thermo Scientific) and exposed to film. The following antibodies were used in this study: Huwe1 (Bethyl, A300–486A, 1:1000), N-myc (Santa Cruz, B8.4.B, 1:500), c-Myc (Cell Signaling, 9402, 1:500), Mcl-1 (Santa Cruz, S-19, 1:200), Miz-1 (Santa Cruz, H-190, 1:200), beta catenin (Cell Signaling, 8480, 1:1000), Erk1/2 (Cell Signaling, 4695, 1:1000), phospo-Erk1/2 (Cell Signaling, D13.14.4E, 1:1000), p53 (Leica, CM5, 1:1000), vinculin (Sigma, hVIN-1, 1:5000) and actin (EMD Millipore, C4, 1:5000).
Horseradish peroxidase conjugated anti mouse igg or anti rabbit igg
Manufactured by GE Healthcare
Horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG is a secondary antibody conjugate used in various immunoassay techniques. It is capable of binding to the primary antibody targeting mouse or rabbit immunoglobulin G (IgG) and is labeled with the enzyme horseradish peroxidase, which can be detected through colorimetric or chemiluminescent reactions.
Automatically generated - may contain errors
Lab products found in correlation
Mcl 1, by Santa Cruz Biotechnology (2 mentions)
Bis tris gel, by Thermo Fisher Scientific (2 mentions)
β catenin, by Cell Signaling Technology (2 mentions)
Supersignal west pico ecl substrate, by Thermo Fisher Scientific (2 mentions)
Huwe1, by Fortis Life Sciences (2 mentions)
N myc, by Santa Cruz Biotechnology (2 mentions)
Miz 1, by Santa Cruz Biotechnology (2 mentions)
Phospo erk1 2, by Cell Signaling Technology (2 mentions)
Hvin 1, by Merck Group (2 mentions)
C myc, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Erk1 2, by Cell Signaling Technology (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Anti nmdar1, by Merck Group (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti actin, by Merck Group (1 mentions)
Anti synaptophysin, by Merck Group (1 mentions)
Imagequant las 4010 system, by GE Healthcare (1 mentions)
Anti gfap, by Merck Group (1 mentions)
Ecl kit, by GE Healthcare (1 mentions)
5 protocols using horseradish peroxidase conjugated anti mouse igg or anti rabbit igg
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Hippocampal Proteins
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Hippocampal slices were lysed in a solution containing 4% SDS, 2 mM EDTA, 1 mM Na3VO4, 10 mM NaF and 50 mM Tris–HCl (pH 6.8). Samples were separated by SDS/PAGE (20 μg protein per sample) and transferred to nitrocellulose membranes using a semi dry blotting apparatus (1.2 mA/cm2; 1 h). The membranes were blocked with 2% albumin in Tris-buffered saline with Tween 20 (T-TBS) and then incubated overnight (4 °C) with anti-PI3K (1:2000, Cell Signaling), anti-Akt (1:2000; Cell Signaling), anti-NMDAR1 (1:5000; Millipore), anti-GFAP (1:5000; Sigma-Aldrich), anti-synaptophysin (1:5000; Millipore), anti-tubulin βIII (1:5000; Abcam), or anti-actin (1:5000; Millipore). β-actin was used as a loading control. Subsequently, the membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (1:10,000; GE Healthcare) for 1 h. The chemiluminescence signal was detected in an Image Quant LAS4010 system (GE Healthcare) using an ECL kit (GE Healthcare). Results are expressed as percentages relative to non-infection control conditions.
3
Immunoprecipitation and Western Blot Protocol
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Cells (5 × 108) in YES were harvested and resuspended in 0.3-Buffer T (25 mM HEPES-KOH (pH 7.5), 0.1 mM ethylenediaminetetraacetic acid (EDTA), 10% glycerol, 0.3 M KCl and 0.1% Tween 20) containing 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (0.35 μg/ml benzamidine, 0.7 μg/ml pepstatin, and 0.5 μg/ml leupeptin). The cells were homogenized with glass beads in a bead shocker (Yasui Kikai) seven times for 60 s at 4°C. The cell extract was centrifuged for 10 min at 15,000 rpm at 4°C. The resultant supernatant was incubated with Dynabeads Pan Mouse IgG (Invitrogen) for 2 h. After incubation, the beads were washed six times with 0.3-Buffer T. The beads were resuspended in loading buffer. Before immunoprecipitation, 50 μl of Dynabeads Pan Mouse IgG (Invitrogen) were equilibrated overnight in 5 ml of phosphate-buffered saline (PBS) containing 1 mg/ml of bovine serum albumin (BSA). The beads were incubated with an antibody for several hours at 4°C.
The immunoprecipitated and input samples were separated by polyacrylamide gel electrophoresis, and the proteins were blotted onto nitrocellulose membranes. The membranes were first probed with primary antibodies, and then incubated with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (GE Healthcare Life Science).
The immunoprecipitated and input samples were separated by polyacrylamide gel electrophoresis, and the proteins were blotted onto nitrocellulose membranes. The membranes were first probed with primary antibodies, and then incubated with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (GE Healthcare Life Science).
4
Western Blot Analysis of Protein Expression
5
Protein Extraction and Western Blot Analysis
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Cells were collected by centrifugation and washed with distilled water. Cell pellets were resuspended in protein extraction buffer (50 mM Tris–HCl, 5 mM EDTA, 150 mM KCl, 10 mM MgCl2, 10% glycerol, 0.2% NP-40, 1 mM dithiothreitol, 20 mM β-glycerophosphate, 0.1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride) containing 1× Complete Protease Inhibitor (Roche), and disrupted with zirconia beads and Multibead Shocker (Yasui kikai, Japan). Cell extracts were separated by SDS-PAGE. An antibody against Atf1 (33 (link)) or tubulin (Sigma, T5168) was used as a primary antibody. Detection was performed with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (GE Healthcare).
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