Western blot analysis was carried out for different HCN proteins (1-4) in the bladder tissue. Briefly, bladder tissue was homogenized using CelLytic™ MT Mammalian Tissue Lysis/Extraction Reagent (Sigma, USA) in the presence of phenylmethylsulfonyl fluoride (1 mM), sodium orthovanadate (2 mM) and protein inhibitor cocktail (Sigma, USA). Protein estimation was done by BCA Protein Assay Kit (Pierce, Rockford, Illinois). An equal amount (50 μg/well) of denatured proteins was loaded in 10% tricine-SDS gel and blotted on polyvinylidene fluoride (PVDF) membranes using wet transfer system. After blocking (2 h at 37°C), membranes were incubated overnight at 4°C with primary antibodies specific for HCN1, HCN4 and Actin-β (Santa Cruz Biotechnology), HCN2 and HCN3 (Abcam, USA), in blocking buffer (pH 7.5). The membranes were then re-incubated for 2 h at room temperature with secondary immunoglobulin G (IgG)-conjugated with horseradish peroxidase (Santa Cruz Biotechnologies, USA). The blots were developed using luminol (Thermo Scientific, USA) and measured on Versa doc imaging system (Model 4000; BioRed, USA). Densitometry for protein specific band was done using AlphaEase FC StandAlone V. 4.0.0 software. β-Actin was used as an internal control to normalize the band density.
Protein inhibitor cocktail
Manufactured by Merck Group
Sourced in United States, Israel, Germany, Japan, China
The Protein Inhibitor Cocktail is a laboratory product designed to inhibit the activity of various proteins. It is a mixture of chemical compounds that can effectively suppress the function of specific proteins in a controlled experimental setting. The core function of this product is to provide a reliable tool for researchers to study the role and behavior of proteins in different biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Gel documentation 2000 system, by Bio-Rad (10 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Ripa buffer, by Cell Signaling Technology (5 mentions)
β actin, by Santa Cruz Biotechnology (5 mentions)
Ripa buffer, by Merck Group (5 mentions)
β actin, by Merck Group (4 mentions)
Sds polyacrylamide gel electrophoresis, by Bio-Rad (4 mentions)
Luminol, by Thermo Fisher Scientific (3 mentions)
Quantity one software, by Bio-Rad (3 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (3 mentions)
Complete mini, by Roche (3 mentions)
Hcn2 and hcn3, by Abcam (2 mentions)
Secondary immunoglobulin g igg conjugated with horseradish peroxidase, by Santa Cruz Biotechnology (2 mentions)
Cellytic mt mammalian tissue lysis extraction reagent, by Merck Group (2 mentions)
Ras10 mouse monoclonal primary antibody, by Merck Group (2 mentions)
Chemidoc xrs hq system, by Bio-Rad (2 mentions)
Pefabloc, by Merck Group (2 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
81 protocols using protein inhibitor cocktail
1
Western Blot Analysis of HCN Proteins
2
Ras Protein Extraction and Western Blot
3
Western Blot Protein Analysis
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Protein extracts were prepared from LCLs whole cell lysates using CelLytic M cell lysis solution and Protein inhibitor cocktail (Sigma). Western blots were prepared in duplicates, using equivalent protein concentrations and Novex NuPAGE Tris-Acetate gel electrophoresis (Invitrogen). Primary antibodies were incubated with the membrane overnight at 4°C in 5% skimmed milk and detected using secondary antibodies (Supplementary Material, Table S2 ). ECL (Amersham) was used for signal detection.
4
Proteome Profiling of CRC Cells and Tissues
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CRC cell line pellet samples (2 × 108 cells per replicate, four replicates per cell line) were resuspended with PBS up to 2 ml and then solubilized by adding 2 ml of ice-cold 2× lysis buffer (1% w/v CHAPS). Tumor and NAT samples (average 568 mg) were cut into small pieces (cubes, ∼3 mm in size), and 5 ml of ice-cold PBS containing protein inhibitor cocktail (Sigma, cat#P8340-5 ml) was added. Tissues were first homogenized twice for 20 s using an Ultra Turrax T25 homogenizer (IKA-Labortechnik) set at a speed of 20,000 rpm and then 20 s using an Ultra Turrax T8 homogenizer (IKA-Labortechnik) set at speed 25,000 rpm. Then, 550 μl of ice-cold 10× lysis buffer (5% w/v CHAPS) was added to each sample. After 60-min incubation with tumbling at 4 °C, tissue samples and CRC cell line samples were spun at 10,000g for 30 min at 4 °C. Supernatants were transferred into tubes containing 1 mg of W6/32 antibody covalently cross-linked protein A magnetic beads, and MAPs were immunoprecipitated as described (30 ). MAP extracts were then dried using a Speed-Vac and kept frozen before MS analyses.
5
SARS-CoV-2 RBD Crosslinking and Immunoprecipitation
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HEK293TN and hACE2-HEK 293 TN cells were grown at 90% confluency in 150 mm plates. Cells were then detached in PBS. After 3 washes, cells were resuspended in PBS buffer containing 20 μg/ml of recombinant SARS-CoV-2-RBD and incubated with agitation at 4 °C for 60’. Primary amine crosslinker Disuccinimidyl Suberate (DSS) (Thermo Fisher Scientific cat no. 21555) was then added to the mix at a 2 mM final concentration. Crosslinking reactions were performed at room temperature (RT) for 30 min in agitation and were then quenched by addition of 20 mM of TRIS pH 7.4 for 15’.20 (link) After two additional washes in PBS, cells were lysed in the following buffer: 50 mM TRIS pH 7.4, 150 mM NaCl, 1% TRITON and 1:100 protein inhibitor cocktail (Sigma–Aldrich cat no. P8340).
1 mg of total cell lysates were then incubated for 2 h at 4οC in constant rotation with protein G Dynabeads (Life technologies cat. No. 10004D) previously incubated with 25 μg/ml of anti-hACE2 antibodies (R&D systems, cat no. AF933). The beads were then washed three times with lysis buffer and proteins were eluted from the beads by incubation at 95 °C for 10 min with 2X Laemmli sample buffer. Immunoprecipitated samples were then loaded on separate gels for silver staining (to control Immunoprecipitation efficiency), coomassie blue staining (for subsequent mass spectrometry analysis) and western blotting.
1 mg of total cell lysates were then incubated for 2 h at 4οC in constant rotation with protein G Dynabeads (Life technologies cat. No. 10004D) previously incubated with 25 μg/ml of anti-hACE2 antibodies (R&D systems, cat no. AF933). The beads were then washed three times with lysis buffer and proteins were eluted from the beads by incubation at 95 °C for 10 min with 2X Laemmli sample buffer. Immunoprecipitated samples were then loaded on separate gels for silver staining (to control Immunoprecipitation efficiency), coomassie blue staining (for subsequent mass spectrometry analysis) and western blotting.
6
Western Blot Analysis of HCN Proteins
7
Western Blot Protein Expression Analysis
8
Cell Lysis and Protein Analysis
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Cells were harvested and washed with PBS at room temperature. Cells were incubated for 40 min in an ice-cold lysis buffer (10 mM Tris-HCl, pH 7.5, 80 mM NaCl, 1% Triton X-100, 1 mM dithiothreitol, 1 mM PMSF, 10 mM NaF, 2 mM Na3VO4 and 10 µg/ml protein inhibitor cocktail, Sigma Aldrich). The lysates were centrifuged at 800×g for 5 min at 4°C and stored at −20°C. The protein content was determined by the Bradford assay. Total cell lysates were analyzed by SDS-PAGE and immunoblotting as described [60] (link) using the antibodies described in table 2 (Table 2 and Table S1 ).
9
Quantifying GAP-43 and ATF-3 in DRG and Sciatic Nerve
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DRG or sciatic samples were homogenized in lysis buffer (1% Triton, 20 mM Tris pH 7.6, 1 mM EDTA, 150 mM NaCl, 1 mM NaF, 1 mM NaVO3) with protein inhibitor cocktail (Sigma) followed by total protein quantification. Proteins were resolved in 12% SDS-PAGE gel and transferred to a nitrocellulose membrane (Bio-Rad). The membrane was then blocked with 5% non-fat milk in TBS-T solution for 1 h, followed by incubation with anti-GAP-43 (sciatic sample, Santa Cruz, RRID:AB_640874 ), anti-ATF-3 (DRG sample, Santa Cruz, RRID:AB_2258513 ) or anti-β-actin/actin primary antibody overnight at 4°C (1:1000, Santa Cruz, RRID:AB_2223228 /Millipore, RRID:AB_2223041 ). On the next day, anti-rabbit secondary antibody was incubated for 1 h and chemiluminescence was measured using a ChemiDoc XRS+ imaging system (Bio-Rad) after development in chemiluminescence reagent (Westar Nova 2011, CYANAGEN). Bands intensity was determined using ImageJ software (NIH, United States).
10
PPARγ and AMPK Signaling Pathway
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Cells were washed twice with ice-cold PBS and subsequently lysed in equal volumes of cell lysis buffer containing protein inhibitor cocktail (Sigma) for 30 min. Protein concentration was measured by BCA protein assay kit (Pierce). The lysates mixed with a certain amount of 5 × loading buffer and boiled at 100 ℃ for 10 min. The protein was separated by 10% SDS-PAGE and transferred to PVDF membrane, blocked in 5% non-fat dried milk dissolved in PBS-T for 1 h and immunoblotted with antibodies specific for PPARγ, FABP4, t-AMPKα, P-AMPKT172, t-ACC, P-ACCS79 and β-actin. Finally, the immunoblots were quantified using the Metamorph software and expressed as a ratio of phosphorylated to total protein or total protein to β-actin.
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