Immunoblot analysis was performed as previously reported12 (link). Total proteins were extracted from ciBAs and the control fibroblasts with RIPA buffer [50 mM Tris–HCl (pH 8.0), 0.15 M sodium chloride, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 1% NP-40 substitute; Wako] and protease inhibitor cocktail (Wako). The proteins were subjected to 10% SDS-PAGE and transferred to a PVDF membrane (Thermo Fisher Scientific, DE, USA). The membranes were blocked with 5% skim milk followed by incubation with UCP1 antibody (MAB6158, R&D Systems, MN, USA) or β-Actin antibody (A5316, Sigma-Aldrich, MO, USA) at 4 °C overnight. Total SMAD2/3 proteins and phosphorylated SMAD2/3 proteins were detected by Smad2/3 (D7G7) XP Rabbit mAb (#8685, Cell Signalling Technology, MA, USA) and Phospho-Smad2 (Ser465/567)/Smad3 (Ser423/425) (D27F4) Rabbit mAb (#8828, Cell Signalling Technology), respectively. The membranes were incubated with either HRP-conjugated anti-rabbit or anti-mouse secondary antibody (Santa Cruz Biotechnology, CA, USA) for 1 hr at room temperature. Immunoreactive bands were detected by Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Darmstadt, Germany). Each band intensity was quantified by densitometry using ImageJ software (National Institutes of Health).
Hrp conjugated anti rabbit or anti mouse secondary antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
HRP-conjugated anti-rabbit or anti-mouse secondary antibodies are laboratory reagents used in immunoassays and other applications that require the detection of primary antibodies. These secondary antibodies are conjugated with horseradish peroxidase (HRP), an enzyme that can be used to catalyze colorimetric or chemiluminescent reactions for signal amplification and visualization.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Santa Cruz Biotechnology (3 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Fujifilm (2 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (2 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Stat3, by Cell Signaling Technology (2 mentions)
P erk, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Np 40 substitute, by Fujifilm (1 mentions)
β actin antibody, by Merck Group (1 mentions)
Bca assay, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Hnrnpa1, by Santa Cruz Biotechnology (1 mentions)
Chemiluminescence method, by Merck Group (1 mentions)
Srsf1, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Phospho s6 s235 236, by Cell Signaling Technology (1 mentions)
Histone h3, by Abcam (1 mentions)
20 protocols using hrp conjugated anti rabbit or anti mouse secondary antibody
1
Immunoblot Analysis of Protein Expression
2
Flag-Tagged TSC22 Nuclear Fractionation and Detection
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The nuclear fraction of Flp-InTMT-RexTM293 cells expressing Flag-TSC-22 was prepared. A whole-cell extract was prepared from Flp-InTMT-RexTM293 cells expressing Flag-TSC22(86). The nuclear or whole-cell extracts were immunoprecipitated using anti-FLAG M2-Agarose and electrophoresed on a 4–20% gradient gel (Multigel II mini 4/20, Cosmo Bio, Tokyo, Japan). After electrophoresis, the blot was prepared by applying an electric current for 7 min using a Bio-Rad Trans-Blot Turbo transfer system and blocked with 2% bovine serum albumin (BSA, Fujifilm, Wako Pure Chemical)-TTBS for 1 h. The blotted membrane was incubated with rabbit anti-human TSC22D1 polyclonal antibody, mouse anti-human histone H1 monoclonal antibody (Genetex, Irvine, CA, USA), and rabbit anti-human GNL3 polyclonal antibody (Genetex) 1000-fold diluted with 2% BSA-TTBS at room temperature for 2 h. Then, washing with TTBS for 10 min was repeated three times, and the blots were incubated with 4000-fold diluted HRP-conjugated anti-rabbit or anti-mouse secondary antibody (Santa Cruz Biotechnology) at room temperature for 1 h, followed by washing with TTBS for 10 min three times and detection using ELC Plus (Thermo Fisher Scientific) and X-ray film (Fuji super RX, Fujifilm, Tokyo, Japan).
3
Western Blot Protein Analysis
4
Western Blotting Procedure for Protein Quantification
5
Western Blot Analysis of PI3K/AKT Pathway
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Adherent cells were first washed with 1× PBS, then thoroughly dried, and consequently frozen down at –20° C overnight. The day after, frozen cells were scraped and lysed with RIPA buffer (150 mM NaCl, 1.0% IGEPAL®, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0. [Sigma], and 1× protease inhibitor cocktail [Roche]). Lysates (20 μg) were resolved by SDS-PAGE and transferred to nitrocellulose or PVDF membranes; these were first incubated with primary antibodies at 4° C overnight, followed by incubation with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (1:10,000) (Santa Cruz Biotechnology) for 1 h at room temperature. Immunoreactive bands were visualized by enhanced chemiluminescence (Thermo Scientific). Antibodies and dilutions: phospho-AKT-S473 (Cell Signaling 9271, 1:500), AKT (Cell Signaling 9272, 1:1500), phospho-PRAS40 (Cell Signaling 2997, 1:500), phospho-S6-S235/236 (Cell Signaling 4858, 1:500), S6 (Cell Signaling 2217, 1:1500), β-Actin (Cell Signaling 4970, 1:5000), SETD2 (Sigma HPA042451, 1:500), Histone H3K36me3 (Active Motif 61101, 1:500), Histone H3 (Abcam ab10799, 1:500), p110α (Cell Signaling 4249, 1:500), p110β (Cell Signaling 3011, 1:500), p110δ (Cell Signaling 34050, 1:500).
6
Immunoblot Analysis of Protein Expression
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For immunoblot analysis, total proteins were extracted from the control fibroblasts, ciBAs, and Capsaicin-treated ciBAs with RIPA buffer (FUJIFILM Wako) including phosphatase inhibitor cocktail (FUJIFILM Wako) and protease inhibitor cocktail (FUJIFILM Wako). The extracted proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis using a 10% gel concentration and transferred to a polyvinylidene fluoride membrane (Thermo Fisher Scientific, MA, USA). The membranes were blocked with 3% skim milk followed by incubation with antibodies against UCP1 (MAB6158, R&D Systems, MN, USA), COX4 (#4850, Cell Signalling Technology, MA, USA), VDAC1 (ab14734, Abcam, Cambridge, UK), or β-Actin (A5316, Sigma-Aldrich) at 4 ˚C overnight. The membranes were incubated with either HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (Santa Cruz Biotechnology, CA, USA) for 1 h at room temperature. Immunoreactive bands were detected by Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Darmstadt, Germany). The intensity of each band was quantified by densitometry using ImageJ software (National Institutes of Health). The experiments were performed independently at least twice.
7
Antibody-Based Protein Detection Protocols
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The primary antibodies used for immunofluorescent staining and immunoblotting-based experiments were as follows: actin (A2066, Sigma), DNA (61,014, Progen Biotechnik GmbH), K48 polyubiquitin (05–1307, EMD Millipore), K63 polyubiquitin (Apu3, 05–1308, EMD Millipore), mono- and polyubiquitinated conjugates monoclonal antibody (FK2, Enzo Lifesciences), Parkin (ab15954, Abcam), phospho-ubiquitin Ser65 (#37642; Cell Signaling), PINK1 (D8G3, Cell Signaling), and TOM20 (D8T4N, Cell Signaling). Detection of primary antibody-binding was performed using anti-mouse Alexa Fluor 555 conjugated antibodies (Life Technologies) or anti-rabbit Alexa Fluor 647 conjugated antibodies (Life Technologies) for immunofluorescent staining experiments and HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (Santa Cruz Biotechnology), as appropriate, for immunoblotting.
8
Quantifying Cyclin D1 and pRB Expression
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Stable cancer cells (NCI-N87, ShScramble, and ShCCND1) were lysed in RIPA buffer including protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA). The protein concentration of cell lysate was determined using the BCA™ protein assay kit (Thermo Scientific, Rockford, IL, USA). Equal amounts of total protein were electrophoresed by 10% SDS-PAGE, transferred onto nitrocellulose membranes, and incubated with antibodies against cyclin D1 (1:500, Santa-Cruz Biotechnology, Santa Cruz, CA, USA), pRB (1:200, Cell Signaling Technology, Beverly, MA, USA), and β-actin (1:500, Santa-Cruz Biotechnology) at 4°C. Membranes were then incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (1:1000, Santa-Cruz Biotechnology). Signals were detected using an ECL Test Kit (KPL, Gaithersburg, MD, USA). β-actin served as the internal standard. Densitometric analysis was performed using ImageJ software.
9
Protein Expression Analysis in Kidney Tissue
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Proteins were extracted from kidney tissues according to standard protocols. In brief, protein samples were separated on 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (40 μg/lane), and then were transferred to a nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% non-fat dry milk in TBS-T buffer, and then incubated with rabbit polyclonal anti-Bax (1:1000; Cell Signaling Technologies [CST], Danvers, MA, USA), Bcl-2 (1:1000; CST, CA), Caspase3 (1:1000; CST, CA), TGF-β1 (1:1000; CST, CA), HMGB1 (1:800; CST, CA), or Smad3 (1:2000; abcam, Cambridge, UK) antibody or mouse monoclonal anti-GAPDH antibody (1:1000; Santa Cruz, Dallas, TX, USA) overnight at 4°C. After extensive rinsing with TBS-T buffer, blots were incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (Santa Cruz) and developed using an enhanced chemiluminescence system (ECL Kit; Pierce Biotechnology Inc., Rockford, IL, USA) and captured on light-sensitive imaging film (Kodak, Tokyo, Japan).
10
Protein Expression and Apoptosis Analysis
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Cells were lysed in RIPA buffer. Resolved proteins were electrophoretically transferred to polyvinylidene fluoride membranes. Specific proteins were sequentially blotted with primary antibodies: SOX4 (Catalogue# ab80261, Abcam, Cambridge, Mass, USA), cleaved caspase-3, cleaved caspase-7, cleaved poly-ADP ribose polymerase (PARP; Cell Signaling Technology, Danvers, MA, USA), X-linked inhibitor of apoptosis protein (XIAP) and polyclonal anti-GAPDH (Santa Cruz Biotechnology, CA, USA). Each membrane was incubated with anti-rabbit or anti-mouse HRP-conjugated secondary antibody (Santa Cruz Biotechnology). Immunoreactive proteins were visualized on the enhanced chemiluminescence detection system HRP substrate (Millipore, Billerica, MA, USA). The immunoreactive bands were quantified by densitometric analysis using the luminescent image analyzer LAS-4000.
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