iPS-CMs were fixed in formalin for 10 min before being washed 3X with PBS. Cells were then permeabilized with PBS containing 0.25% Triton X-100. After permeabilization, cells were incubated in a blocking buffer (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h before being incubated with anti-troponin I (MilliporeSigma), Anti-Sarcomeric Myosin (Developmental Studies Hybridoma Bank, Iowa City, IA, USA), and anti-alpha actinin (MilliporeSigma) overnight according to manufacturer’s instructions. Coverslips were then washed 3X in PBS before being incubated with Donkey anti-Mouse Alexa Fluor 488 (Thermo Fisher) for an hour before being washed 3X in PBS and stained with Dapi (MilliporeSigma). Coverslips were then imaged using an EVOS cell imaging system.
Blocking buffer
Manufactured by Santa Cruz Biotechnology
Sourced in United States, Sweden
Blocking buffer is a laboratory reagent used to prevent non-specific binding in immunoassays and other protein-based experiments. It contains a mixture of proteins and surfactants that block unoccupied binding sites on the solid support or membrane, reducing background signal and improving the specificity of the assay.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 donkey anti mouse, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Protein extraction reagent, by Beyotime (1 mentions)
Horseradish peroxidase, by Abcam (1 mentions)
P enos, by Cell Signaling Technology (1 mentions)
Cell lysis buffer system, by Santa Cruz Biotechnology (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
Rock2, by Cell Signaling Technology (1 mentions)
Tnf α, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Interleukin 6 (il 6), by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
Histone h3, by Abcam (1 mentions)
Nf κb, by Abcam (1 mentions)
Active caspase 3, by Abcam (1 mentions)
Ecl kit, by Cytiva (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Corresponding secondary antibody, by Abcam (1 mentions)
Caspase 12, by Abcam (1 mentions)
5 protocols using blocking buffer
1
Immunocytochemistry of Induced Pluripotent Stem Cell-Derived Cardiomyocytes
2
Protein Expression Analysis in HPMVECs
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Cultured HPMVECs were homogenized with the cell lysis buffer system (Santa Cruz) on dry ice. The protein was extracted using protein extraction reagents (Beyotime) per the manufacturer’s instructions. After centrifugation of homogenates, the concentration of protein was measured with BCA method by using a BCA assay kit (Invitrogen). Protein samples were separated by SDS-PAGE and then transferred to the PVDF membranes electronically. The membranes were then incubated with the blocking buffer (Santa Cruz) to eliminate the non-specific binding sites. Then, the membranes were incubated with primary antibodies against ROCK2 (Cell Signaling Tech, 1: 2000), eNOS, (Cell Signaling Tech, 1: 2000) p-eNOS (Cell Signaling Tech, 1: 2000), Bcl2 (Santa Cruz, 1: 2000), active caspase3 (Abcam, 1: 2000), NF-κB (Abcam, 1: 1000), IL-6 (Abcam, 1: 2000), TNF-α (Abcam, 1: 1000), GAPDH (Abcam, 1: 5000), and Histone-H3 (Abcam, 1: 2000) at 4°C for 12 h. After washing in TBST, membranes were further incubated with secondary antibodies conjugated with horseradish peroxidase (Abcam) at room temperature for 2 h. The immunoblots were visualized with an enhanced chemiluminescence Western blotting detection system (ECL, Fisher Scientific).
3
Western Blot Analysis of Breast Cancer Cell Lines
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Protein extracts from seven breast cancer cell lines were obtained using a lysis buffer (10 mM Tris-HCl (pH 7.4); 150 mM NaCl, 0.1% NP-40; 5 mM EDTA; 50 mM NaF) in the presence of protease inhibitors. Total cellular proteins (50 and 100 μg for CDKN2A and MTAP analysis, respectively) were separated on 12% SDS-polyacrylamide gels and electro-transferred onto nitrocellulose or polyvinylidene fluoride (PVDF) membranes (Protran, Schleicher & Schuell, Germany). Immunoblotting was carried out with rabbit anti-human p16 (CDKN2A) (C-20)-G polyclonal antibody, goat anti-MTAP polyclonal antibody (N-20) and rabbit anti-β-tubulin (H-235) polyclonal antibody (dilution 1:200, 1:100 and 1:500, respectively), purchased from Santa Cruz Biotechnology Inc. (Dallas, Texas, USA). Membranes were then reacted with secondary antibodies (dilution 1:3000 in blocking buffer, Santa Cruz Biotechnology) and developed using the ECL Kit (Amersham Biosciences, Sweden).
4
Western Blotting Analysis of UPR Markers
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Cells were collected and suspended in a RIPA lysis buffer system (Santa Cruz) and whole-cell extract was prepared. Total protein was extracted using the Protein Extraction kit (Beyotime) according to the instructions provided by the manufacturer. A BCA kit (Beyotime) was used to measure the protein concentrations. The protein was separated by vertical SDS-PAGE and then transferred to the PVDF membranes electronically. The blocking buffer (Santa Cruz) was applied to the membranes. Primary antibodies against GRP78 (1: 2000; Abcam), PERK (1: 2000; CST), p-PERK (1: 2000; CST), cleaved ATF6 (1: 2000; Abcam), IRE-1α (1: 4000; CST), p-IRE-1α (1: 2000; CST), CHOP (1: 2000; Abcam), caspase12 (1: 2000; Abcam), and GAPDH (1: 4000; Sigma) were used to incubate the membranes at 4°C for 8 h in TBST. Then, the corresponding secondary antibodies (Abcam) were used to incubate the membranes at room temperature for 1 h. The membranes were developed by an ECL kit (pierce) and then exposed with the Gene Genius system (Syngene). The densities of the blots were then analyzed with Image J software.
5
Protein Expression Analysis of Pancreatic Tissue
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The harvested pancreatic tissue was lysed on ice using a lysis buffer system (Santa Cruz) supplemented with PMSF (Santa Cruz). Total protein was extracted by using a Total Protein Extraction Kit (Beyotime). Nuclear protein was extracted by using a Nuclear Protein Extraction Kit (Beyotime). The protein concentration was measured with a BCA kit (Peirce). The protein was separated after being subjected to SDS-PAGE. Then, the separated proteins were transferred to PVDF membranes electronically. The membranes were then incubated with blocking buffer (Santa Cruz). Primary antibodies against TRAF1, MKK4, phosphorylated MKK4, MKK7, phosphorylated MKK7, JNK, phosphorylated JNK, NF-κB, IL6, TNFα, GAPDH, and Histone H3 were used to incubate the membranes at 4°C for 8 h. Then, the membranes were washed in TBST and further incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. The immunoblots were developed by Lumi-Light substrate (Pierce) and visualized on X-ray films.
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