Protein extractions from cultured cells with a modified buffer from cultured cells were followed by immunoprecipitation and immunoblotting assays with the corresponding antibodies, as described in the following. The detection of SAK-HV was determined by immunoblotting using an antibody purchased from CWbio, while the co-immunoprecipitated proteins were detected using antibodies against Uba1 (Abcam plc), HSP105 (Abcam plc), HSP70 (Abcam plc), and IDE (Santa Cruz Biotechnology). Immunoprecipitated material was detected using the indicated primary antibody and HRP-conjugated anti-rabbit or anti-mouse IgG (Sigma-Aldrich).
Hrp conjugated anti mouse or anti rabbit igg
Manufactured by Merck Group
Sourced in United States, Macao
HRP-conjugated anti-mouse or anti-rabbit IgG is a laboratory reagent used in various immunoassays and detection techniques. It consists of an anti-mouse or anti-rabbit immunoglobulin G (IgG) antibody covalently linked to the enzyme horseradish peroxidase (HRP). This conjugate can be used to detect and quantify the presence of mouse or rabbit antibodies in biological samples.
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Lab products found in correlation
Bovine serum albumin (bsa), by Thermo Fisher Scientific (5 mentions)
Ripa lysis buffer, by Merck Group (4 mentions)
Protease inhibitor, by Roche (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Phosphatase inhibitor, by Roche (3 mentions)
X omat film, by Kodak (3 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (2 mentions)
Hsp70, by Abcam (1 mentions)
Skim milk, by Merck Group (1 mentions)
Immobilin western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Protease phosphatase inhibitor cocktail, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Protease inhibitor tablet, by Roche (1 mentions)
Mini beadbeater 16, by Biospec (1 mentions)
Las 500 chemiluminescence imager, by GE Healthcare (1 mentions)
Gapdh, by Merck Group (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
12 protocols using hrp conjugated anti mouse or anti rabbit igg
1
Protein Interactome Analysis by IP-Western
2
Quantitative Protein Analysis by Western Blot
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Total proteins were isolated using RIPA Buffer (Cell Signaling Technology) and Protease/Phosphatase Inhibitor Cocktail (Cell Signaling Technology), followed by polyacrylamide gel electrophoresis on NuPAGE 4–12% Bis-Tris Protein Gels (Thermofisher) using 10–20 μg of protein. Gels were transferred to the PVDF membrane (GE Healthcare) and blocked with 0.1% TBST solution containing 5% skim milk (Difco) for 1 h. Membranes were incubated with 0.1% TBST solution containing 5% BSA and primary antibodies for 1 h, then treated with 0.1% TBST solution containing 5% skim milk for 1 h. HRP conjugated anti-rabbit or anti-mouse IgG (Sigma) was used as a secondary antibody. Membranes were incubated with Immobilin Western Chemiluminescent HRP Substrate (Millipore) for 1 min and developed on X-ray film (AGFA).
3
Cell Lysis and Immunoblotting Protocol
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WCE of one hundred OD600 units of asynchronized or synchronized cells were prepared by glass bead beating (Mini-Beadbeater-16, Biospec,USA) in lysis buffer {(50 mM HEPES/KOH pH7.4, 150 mM NaCl, 1 mM EDTA, 10% Glycerol, 1 mM DTT, 1 mM PMSF, Protease inhibitor tablets (EDTA free, Roche)}. Protein samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with the antibodies specifically indicated in each figure. Antibodies used in this study are as follows: mouse anti-FLAG M2-specific monoclonal antibody (1:1000, Sigma), rabbit polyclonal anti-GFP (1:500, GeneScript), mouse anti-HA 16B12 (1:1000, Millipore), anti-ac-Smc3, polyclonal anti-Smc3 (1:1000), polyclonal anti-Scc1 (1:1000). HRP-conjugated anti-rabbit or anti-mouse IgG was used as the secondary antibody (1:10000, Sigma).
4
Quantitative Immunoblot Analysis of CRISPR-Edited Cells
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Infected PuroR CRISPR-3T3 cells as described above were collected in RIPA lysis buffer (150 mM sodium chloride, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris [pH 8.0]) supplemented with protease inhibitors and PMSF. Protein concentration of the total protein lysate was quantified using Bradford assay (Bio-rad, Hercules, CA) and 40 μg were separated using 4–15% gradient SDS-PAGE and transferred to polyvinylidene fluoride membranes, followed by incubation with antibody to Cas9 (7A9-3A3, Cell Signaling Technology), ORF73 (mLANA) [37 (link)], GAPDH (Sigma-Aldrich, St. Louis, MO). Detection was performed with HRP-conjugated anti-mouse or anti-rabbit IgG (Sigma) by enhanced chemiluminescence reagent (ECL, Thermo Scientific). Data was collected with LAS 500 Chemiluminescence Imager (GE Healthcare).
5
Immunoblotting Protocol for Protein Analysis
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For immunoblotting, TAs or cultured cells were prepared and homogenized in RIPA lysis buffer (Sigma Aldrich, MO) containing a protease inhibitor (Roche, Basel, Switzerland) and phosphatase inhibitor (Roche, Basel, Switzerland) using Bullet Blender Storm (Next Advance, NY). Equal amounts of protein were then separated by 10% SDS-polyacrylamide gel and electrophoretically transferred to polyvinylidene fluoride membranes (Millipore, MA). The membranes were blocked with 5% BSA (Gibco, MA) in TBST (1 M Tris-HCl, pH 7.4, 0.9% NaCl and 0.1% Tween-20) for 1 h and probed with antibodies diluted in 3% BSA blocking solution overnight at 4°C. Membranes were then incubated with HRP-conjugated anti-mouse or anti-rabbit IgG (1:100,000; Sigma Aldrich, MO) for 1 h, and the protein bands were visualized with the enhanced chemiluminescence system (Millipore, MA). Antibodies used in this study are listed in Supplementary Table S1B .
6
T-cell Signaling Pathway Analysis
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Jurkat T cells were stimulated with T. gondii or LPS with or without pretreatment with specific inhibitors. At the indicated time points, cells were washed with phosphate-buffered saline (PBS), and proteins were extracted using PRO-PREPTM Protein Extraction Solution (iNtRON Biotechnology, Seoul, Korea) for 15 min on ice. After centrifugation at 4°C for 15 min at 14,000 g, the supernatants were collected, and equal amounts of protein from each sample were resolved by standard SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline (20 mM Tris, 137 mM NaCl, pH 7.6) containing 0.1% Tween 20 (TBST). After washing once in TBST, the membranes were incubated overnight at 4°C with anti-p-AKT, anti-AKT, anti-p-ERK1/2, anti-ERK1/2, anti-p38 MAPK, anti-p-p38 MAPK, anti-p-JNK1/2, anti-JNK1/2, and α-tubulin antibodies (all from Cell Signaling Technology) diluted in 5% bovine serum albumin in TBST. Following 3 washes in TBST, the membranes were incubated for 120 min with HRP-conjugated anti-mouse or anti-rabbit IgG (Sigma-Aldrich) diluted at 1:10,000 in incubation buffer. After extensive washing, the bound antibodies were visualized by enhanced chemiluminescence (Amersham Biosciences, Freiburg, Germany).
7
Filaggrin Protein Expression in HaCaT Cells
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HaCaT cells were plated in 100mm culture dishes. Twenty-four hours later, the cells were treated with 0.25, 0.5, 1, 2 mg/mL of ACTPER for 72 h. After treatment, these same cells were washed with cold PBS and lysed using a phosphosafe extraction buffer (Novagen, Madison, WI, USA). Total protein contents in the cell lysates were determined by a Bradford assay kit (Abcam, Cambridge, UK) according to the manufacturer’s protocol. After reconstituting in the sample buffer, 10 micrograms of protein samples were subjected to SDS-PAGE on Bolt™ 10% Bis-Tris Plus Gels (ThermoFisher, Waltham, MA, USA), and electrophoretically transferred to PVDF membranes (Millipore, Burlington, MA, USA). The membranes were reacted with primary antibodies against filament aggregating protein (Filaggrin) (sc-80609, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-actin (A5441, 1:5000, Sigma). Membranes were then incubated with HRP-conjugated anti-mouse or anti-rabbit IgG (1:100,000, Sigma) and visualized in films using ECL solution (Millipore, Billerica, MA, USA).
8
Western Blot Analysis of Protein Expression
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RWPE-1 cells or THP-1 macrophages were washed with cold PBS lysed with RIPA lysis buffer (Sigma-Aldrich, MO) containing a protease inhibitor (Roche, Basel, Switzerland) and a phosphatase inhibitor (Roche, Basel, Switzerland). Equal amounts of protein were then separated by 10% SDS-polyacrylamide gel and electrophoretically transferred to PVDF membranes (Millipore, MA, USA). The membranes were blocked with 5% BSA (Gibco, MA) in TBST (1 M Tris-HCl [pH 7.4], 0.9% NaCl, and 0.1% Tween 20) for 1 h and incubated with primary antibodies diluted in a 3% BSA blocking solution overnight at 4 °C. Membranes were then treated with HRP-conjugated anti-mouse or anti-rabbit IgG (1: 100,000; Sigma-Aldrich, MO) for 1 h, and protein bands were visualized with an ECL (Millipore, MA, USA) and X-Omat film (Kodak, Rochester, NY).
9
Western Blot Analysis of Schwann Cells
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For Western blot analysis, sciatic nerves and primary Schwann cells were prepared using RIPA buffer containing a protease inhibitor (Roche), phosphatase inhibitor cocktail (Roche) and 1 mM PMSF. Equal amounts of protein samples were subjected to SDS-PAGE on 10% polyacrylamide gels, and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 0.1% TBST containing 1% (w/v) BSA (Invitrogen, Carlsbad, CA), and incubated with primary antibodies diluted in a 3% BSA blocking solution overnight at 4 °C. Membranes were then incubated with HRP-conjugated anti-mouse or anti-rabbit IgG (1:100,000; Sigma) for 1 hr, and protein bands were visualized with ECL (Millipore, Billerica, MA, USA) and X-Omat film (Kodak, Rochester, NY).
10
Western Blot Analysis of LNCaP Cells
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After treatment with TP and HX109, LNCaP cells were washed with cold PBS lysed with radioimmunoprecitation(RIPA) lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) containing a protease inhibitor (Roche, Basel, Switzerland) and a phosphatase inhibitor (Roche, Basel, Switzerland). Equal amounts of protein were then separated by 10% SDS-polyacrylamide gel and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA). The membranes were blocked with 5% bovine serum albumin (Gibco, Waltham, MA, USA) in TBST buffer (1 M Tris-HCl (pH 7.4), 0.9% NaCl, and 0.1% Tween 20) for 1 h and incubated with primary antibodies diluted in a 3% BSA blocking solution overnight at 4 °C. Membranes were then treated with HRP-conjugated anti-mouse or anti-rabbit IgG (1:100,000; Sigma-Aldrich, St. Louis, MO, USA) for 1 h, and protein bands were visualized with an enhanced chemiluminescence solution (Millipore, Burlington, MA, USA) and X-Omat film (Kodak, Rochester, NY, USA).
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