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17 protocols using horseradish peroxidase conjugated anti rabbit antibody

1

Immunohistochemical Quantification of GRO-β

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For immunohistochemistry (IHC) analysis, the TMA sections were deparaffinized in 100% xylene and rehydrated in graded ethanol solutions. The sections were then boiled under pressure in citrate buffer (pH 6.0) for 5 minutes for antigen retrieval. TMA sections were incubated overnight with a primary anti-GRO-β antibody (Catalog 500-P104, PeproTech, Rocky Hill, NJ, USA) diluted 1 : 400 in TBS containing 1% bovine serum albumin. After washing, sections were incubated with anti-rabbit horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). GRO-β immunostaining was evaluated independently by two trained pathologists who were blinded to the clinical background of the cases. Positivity of cell staining was recorded as a percentage (0–100%).
The cutoff point for the GRO-β expression score that was statistically significant in terms of overall survival (OS) was determined using the X-tile software program (Rimm Lab, Yale University, New Haven, CT, USA), as described elsewhere [20 (link)]. The degree of staining was quantified using a two-level grading system, and staining scores were defined as follows: for GRO-β, 0–75 was regarded as low expression while 76–100 was regarded as high expression.
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2

Carnosol Regulation of Smad Signaling

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Cells were treated without or with carnosol and collected at specific time points and lysed in cell lysis buffer (10 mM Tris–HCl, pH 7.4, 150 mM sodium chloride, 1% NP-40, 0.1% SDS, 1 mM EDTA, 1 mM phenyl-methylsulfonyl fluoride, 1 mM NaF, 1 mM Na3VO4), supplemented with protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). 30 μg of protein was separated on 8%–12% NuPAGE Novex Bis-Tris gel systems (Thermo Fisher Scientific, Roskilde, Germany) followed by transfer to PVDF membrane (Millipore, Bedford, MA, USA). The membrane was blocked and probed with antibodies (dil 1:1,000) and incubated with secondary anti-rabbit horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology, Aarhus, Denmark). Antibodies for Smad1/5/8 (total or phosphor) and β-Actin purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). Quantification of Western blots was performed with ImageJ program.
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3

Western Blot Analysis of Signaling Proteins

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Western blot analysis was performed as described previously26 (link). The cells were washed in cold PBS twice and lysed in RIPA buffer (Thermo-Fisher Scientific, Roskilde, Denmark) supplemented with protease inhibitors (Roche, Hvidovre, Denmark) for 30 min in cold room. Samples were centrifuged at 12,000 r.p.m., 4 °C for 10 min. Protein concentrations were determined with a BCA kit (Thermo-Fisher Scientific, Roskilde, Denmark), and equal amounts (30 µg) of proteins were loaded on a polyacrylamide gel (Thermo-Fisher Scientific, Roskilde, Denmark). Blotted nitrocellulose membranes were incubated overnight with primary antibody at 4 °C, and were developed after 1 h incubation with secondary anti-rabbit horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology, Heidelberg, Germany) using an ECL Western blotting kit (GE Healthcare, Brøndby, Denmark) and Kodak films. Antibodies for KIAA1199 was purchased from ProteinTech, Manchester, UK; Antibodies (total or phosphor) specific for CFL1, LIMK1, p38, GSK3β and beta-catanin were obtained from Cell Signaling (Herlev, Denmark); antibodies for DSTN and actin were bought from Sigma-Aldrich (Copenhagen, Denmark). All antibodies were used at 1:1000 dilutions except actin antibodies (1:2500).
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4

Western Blot Protein Analysis

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For Western blot analysis, we used whole cell lysates. The cells were washed in PBS and lysed in RIPA buffer (Thermo Fisher Scientific) supplemented with a protease inhibitor (Roche, Switzerland). Samples were centrifuged for 10 min at 13,000 rpm (4°C). Protein concentration was determined with a BCA kit (Thermo Fisher Scientific), and equal amounts of proteins were loaded on a polyacrylamide gel (Thermo Fisher Scientific). Blotted nitrocellulose membranes were incubated overnight with HA-tag primary antibody (Santa Cruz Biotechnology). The blots were developed after 1-hour incubation with secondary anti-rabbit horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology) using an ECL Western blotting kit (Thermo Fisher Scientific) and Kodak films.
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5

Protein Expression Analysis in SH-SY5Y Cells

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SH-SY5Y cells were lysed with RIPA Lysis Buffer (P0013B; Beyotime, China). The concentration of total protein was measured by protein assay kit (AR0146; Boster, China). The protein samples were separated using 10% or 12% SDS-PAGE gel and blotted to PVDF membranes. Subsequently, the PVDF membranes were incubated overnight at 4℃ in primary antibodies as follows: p-ERK (1:1000; #4370, CST, U.S.), ERK (1:1000; ab184699, Abcam, U.K.), p-Drp1S616 (1:1000; #3455, CST, U.S.), Drp1 (1:1000; #8570S, CST, U.S.), Mfn2 (1:1000; #9482S, CST, U.S.), Mfn1 (1:1000; #14,739, CST, U.S.), Opa1 (1:1000; #67589S, CST, U.S.), LC3B (1:2000; ab192890, Abcam, U.K.), Beclin1 (1:2000; 11,306, Proteintech, China), p62 (1:1000; #5114, CST, U.S.), GAPDH (1:1000; #5174, CST, U.S.). After being washed with Tris-buffered saline, the membranes were incubated in anti-rabbit horseradish peroxidase-conjugated antibodies (1:10000; Santa Cruz Biotechnology, U.S.) for 1 h at room temperature. The target protein bands were detected with Odyssey imaging system (LI-COR, U.S.).
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6

Vastus Lateralis Protein Analysis

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Vastus lateralis biopsies obtained baseline and 6 months were used for the immunoblotting. Samples were homogenized in 1 ml of cold MSD-Tris lysis buffer (Mesoscale Discovery, R60TX-3) supplemented with phosphatase inhibitor and protease inhibitor cocktail tablets, using a FastPrep 24 instrument (MP Biomedicals). Protein concentration was determined by BCA assay kit (Thermo Scientific). 20 micrograms of purified proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on either a 4–20% or a 4–15% gradient gel (BioRad) and transferred onto PVDF membranes (Thermo Fisher Scientific). Membranes were incubated overnight at 4 °C with the following primary antibodies: MEF2A (Thermo Fisher Scientific, PA5–27380), and LAMP2 (Thermo Fisher Scientific #PA1–655). Anti-rabbit horseradish peroxidase-conjugated antibodies (Santa Cruz) were used as secondary antibodies. Densitometric analyses of western blot images were performed by using Image Studio Lite v5.2 (LiCOR) and normalized to Revert Total Protein Stain (LiCOR; #926–11011). Because muscle samples were used up for the MPS and gene expression analyses, only 4–7 subjects per group had remaining samples available for immunoblotting.
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7

Adenoviral Silencing of Calcineurin in Cardiomyocytes

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The Axopatch 700B patch clamp amplifier and Digidata 1322 data converter were from Axon (USA). The Sutter P-97 microelectrode puller was from Sutter (USA) and the BJ-40 glass microelectrode was from Beijing Zhengtianyi Electronics (China). Trypsin and collagenase (type II) were purchased from Sigma (USA). High-glucose Dulbecco’s modified Eagle’s medium (DMEM), premium fetal bovine serum (FBS), phenylephrine (PE), and 5-bromo-2-deoxyuridine (5-BrdU) were purchased from Gibco-BRL (USA). The following antibodies were used: rabbit anti-calcineurin Aβ (CnAβ) antibody (Merck Millipore, Germany), α striated muscle sarcomere actin (α-SCA) antibody (Sigma), horseradish peroxidase-conjugated anti-rabbit antibody (Santa Cruz, USA), rabbit anti-rat Kv4.2 antibody (Abcam, USA), and mouse-derived anti-rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Shanghai Kangcheng Biotech, China). The Genomic DNA Purification Kit was purchased from Fermentas (Canada). The RNeasy Mini Kit was purchased from Qiagen (China). Ad-CnAβshRNA, the gene mediated by the recombinant adenovirus shRNA interference vector for silencing the A subunit β subtype of CaN, and the empty viral vector (null) were prepared by HanBio (Shanghai, China).
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8

Evaluating EMT Markers in Hypoxia

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Anti-E-cadherin antibody, anti-N-cadherin antibody, anti-Snail antibody, anti-HIF-1α antibody, anti-Notch1 antibody, anti-β-actin antibody and horseradish peroxidase-conjugated anti-rabbit antibody, and N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) were purchased from Santa Cruz Biotechnology, Inc., (Dallas, TX, USA). CoCl2 was purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany).
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9

Enzyme-linked Immunoassay for Toxin Detection

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The FP59 variants were coated (75 μL, 2.67 ng/μL in phosphate-buffered saline (PBS) in 96-well EIA/RIA plates (Corning Incorporated, Corning, NY)) for 2 h, blocked with 5% dry milk powder solution in PBS for 30 min and incubated with 60 μL of various dilutions of either anti-Pseudomonas exotoxin A polyclonal rabbit antibody (Sigma, St. Louis, MO) or anti-LF polyclonal rabbit antibody (raised in this laboratory against full length LF) for 1 h. Secondary horse-radish peroxidase-conjugated anti-rabbit antibody (final concentration 0.2 μg/mL 1:2000, 60 μL in PBS, 1 h, Santa Cruz Biotechnology, Dallas, TX) was added and quantified by a colorimetric reaction using 100 μL of substrate solution (40 mM citric acid, pH 3.95, 0.2 g/L 3,3′5,5′ tetramethylbenzidine, 0.1 g/L H2O2). The reaction was stopped with 50 μL of 2 M H2SO4 after 5 min and the absorbance read at 450 nm and 492 nm as reference.
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10

Western Blot Analysis of HDAC1 and TCF-12

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Cells were lysed in a lysis buffer. Protein concentration was determined using the BCA Protein Assay kit (Pierce). Proteins (30μg) were subjected to 10%–12% SDS polyacrylamide gel electrophoresis and subsequently transferred onto Hybond ECL membranes (Amersham). After washing with 0.1% TBS-T, membranes were incubated for 1 h at room temperature in blocking buffer (5% skimmed milk in TBS-T) and then incubated with appropriate antibodies (1:500 dilution; rabbit anti-human HDAC1, rabbit anti-human TCF-12 antibodies are from Cell Signaling, Danver, MA; others are from Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. After washing with TBS-T, membranes were reacted with horseradish-peroxidase-conjugated anti-rabbit antibody (1:2000 dilution, Santa Cruz) for 2 h at room temperature. After further washing with TBS-T, immunoreactive proteins were detected using the Super Signal West Pico Chemiluminescent Substrate (Pierce, Woburn, MA). The efficiency of siRNA and overexpression of HDAC1 and TCF-12 by western-blot also provided in supplementary Figure 1C.
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