H4K20me1, me2, and me3 levels were detected in E14.5 embryonic brain tissue from HET x HET offspring using western blotting. Histone protein extraction was performed using a Histone Extraction Kit (Abcam; Cambridge, United Kingdom) according to the manufacturer’s instructions. Protein extracts were separated on a Mini Trans-Blot Cell (Bio-Rad; Hercules, CA) system using 4–20% Mini-PROTEAN TGX Gels (Bio-Rad; Hercules, CA), transferred using the Trans-Blot® Turbo™ Transfer System (Bio-Rad; Hercules, CA) using pre-assembled Trans-Blot Turbo Mini 0.2 μm PVDF Transfer Packs (Bio-Rad; Hercules, CA), and blocked with EveryBlot Blocking Buffer (Bio-Rad; Hercules, CA). Protein membranes were incubated with rabbit polyclonal anti-H4K20me2 (ab9052) and anti-H4 (ab10158) primary antibodies (Abcam; Cambridge, United Kingdom), washed in TBS buffer (Thermo Scientific; Waltham, MA) with 0.1% Tween-20 followed by a Goat Anti-Rabbit IgG H&L (HRP) (ab205718) secondary antibody (Abcam; Cambridge, United Kingdom). The blots were developed using Clarity™ Western ECL Substrate (Bio-Rad; Hercules, CA). The Precision Plus Protein™ WesternC™ Standard (Bio-Rad; Hercules, CA) was used as a molecular weight marker. Membranes were visualized using a ChemiDoc™ XRS+ System (Bio-Rad; Hercules, CA).
Trans blot turbo mini 0.2 μm pvdf transfer packs
Manufactured by Bio-Rad
The Trans-Blot Turbo Mini 0.2 μm PVDF Transfer Packs are a lab equipment product designed for protein transfer. It utilizes 0.2 μm PVDF membranes to facilitate the transfer of proteins from polyacrylamide gels to a membrane for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Histone extraction kit, by Abcam (1 mentions)
Goat anti rabbit igg h l hrp ab205718 secondary antibody, by Abcam (1 mentions)
Precision plus protein westernc standard, by Bio-Rad (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Mini trans blot cell, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Everyblot blocking buffer, by Bio-Rad (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Intercept pbs blocking buffer, by LI COR (1 mentions)
Odyssey clx fluorescence imaging system, by LI COR (1 mentions)
Trans blot turbo system, by Bio-Rad (1 mentions)
Pageblue protein staining solution, by Thermo Fisher Scientific (1 mentions)
Tbs blotto a, by Santa Cruz Biotechnology (1 mentions)
Sample reducing agent, by Thermo Fisher Scientific (1 mentions)
Lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
5 protocols using trans blot turbo mini 0.2 μm pvdf transfer packs
1
Quantifying Histone H4K20 Methylation
2
Western Blotting of Canine Plasma Proteins
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Canine plasma corresponding to 200–500 μg of total protein was denatured with 1:10 β-mercaptoethanol (Sigma, Cat#M3148) in BoltTM 4x LDS Sample Buffer (Novex life technologies, Cat#B0007) for 5 min at 95°C. Proteins were separated on an acrylamide gel (Bolt 10% Bis-Tris Plus, Invitrogen, Cat#NW00100BOX) and blotted onto a polyvinylidene fluoride (PVDF) membrane (Trans-Blot Turbo Mini 0.2 μm PVDF Transfer Packs, BioRad, Cat#1704156). After blocking for 3 h in 5% Bovine Serum Albumin (BSA; Sigma, Cat#A3059), in 0.2% TTBS (Tween 20, Sigma, Cat#P9416; Tris-Buffered Saline 20x solution ultrapure, Thermo Scientific, Cat#J75892) or in 5% milk (Rapilait, Migros) in TTBS, the membrane was incubated over night at 4°C with the primary antibodies (anti-human REG1A and REG3A) at a 1:1000 dilution in 2% BSA-TTBS or milk-TTBS, and for 2 h with a secondary anti-IgG horseradish peroxidase-coupled antibody (Dako Cat# P0141; Cell Signaling Technology Cat# 7074, RRID:AB_2099233) at a 1:5000 and 1:2000 dilution, respectively. Bands were detected using a Vilber Fusion FX (Witec AG) after 5 min incubation with the chemiluminescent substrate (SuperSignal West Pico PLUS Chemiluminescent Substrate, Thermo Scientific, Cat#34577).
3
Western Blot Protein Analysis Protocol
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Western blot was performed using 4–20% mini-protean tgx stain-free gels, followed by transfer with Bio-Rad trans-blot turbo system using trans-blot turbo mini 0.2 μm PVDF transfer packs. Membranes were blocked for 1 h with intercept (PBS) blocking buffer from Li-Cor (Lincoln, NE, USA), followed by 3× washing with TBS with 0.1% tween. Membranes were incubated with primary antibodies overnight at 4°C, followed by 3× washing with TBS with 0.1% tween and incubation for 1 h at room temperature with Li-Cor secondary antibody (diluted 1:15,000 in PBS). Membranes were scanned using the Odyssey CLx fluorescence imaging system (Li-Cor).
4
Verification of Purified Human CA VI
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Purified protein was verified by SDS polyacrylamide gel (SDS-PAGE) and Western blotting. Purified human CA VI (0.5 μg) in 20 μL running buffer, 10 μL human saliva, and 10 μL human breast milk were run on Any kD Mini-PROTEAN TGX Stain-Free Protein (Bio-Rad, Hecules, CA, USA) gels, 200 V for 30 min. One gel was stained with PageBlue Protein Staining Solution (Thermo Fisher, Waltham, MA). The other gel was transferred to Trans-Blot Turbo Mini 0.2 μm PVDF Transfer Packs (Bio-Rad) membrane with BioRad’s Trans-Blot Turbo Transfer System (3 min). The membrane was blocked with TBS Blotto A (Santa Cruz Biotechnology, Dallas, TX, USA) for 30 min at room temperature. Rabbit anti-human CA VI serum [42 ] or normal rabbit serum (control), both diluted 1:1000, was incubated overnight at 4 °C. The secondary anti-rabbit IgG (#R1364HRP, Acris antibodies, Rockville, MD, USA) diluted 1:20,000 was incubated for 45 min at room temperature. After both incubations, the membrane was washed four times. The Western blot was visualized with WesternBright (Advansta, San Jose, CA, USA) according to the manufacturer’s instructions.
5
Protein Extraction and Western Blot Analysis
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Total protein from SH-SY5Y cells was exacted using a 1× TNE buffer [50 mm Tris–HCl (pH 7.4); 100 mM NaCl. 0.1; mM EDTA) plus 1% (v/v) Triton X-100 (Cat# T8787, Sigma-Aldrich) and protease inhibitor (Cat# P8340, Sigma-Aldrich). Lysates were kept in an orbital shaker incubator at 220 rpm at 4°C for 30 min and then centrifuged for 15 min at 13,000 g at 4°C. The supernatants were transferred to tubes and quantified by DC Protein Assay (Cat# 5000116, Bio-Rad, Milan, Italy). Subsequently, protein samples (60 μg of total protein) were heated at 70°C for 10 min in 1× LDS Sample Buffer (Cat# B0007, Life Technology) plus 1× sample reducing agent (Cat# B0009, Life Technology) and loaded on 10% Bis-Tris Protein Gels (Cat# NW00102BOX, Life Technology) and then transferred the membrane using Trans-Blot Turbo Mini 0.2 μm PVDF Transfer Packs (Cat# 1704156 Bio-Rad). The primary antibodies used were: (a) rabbit anti-TH (Cat# NB300-109, Novus Biologicals) and (b) an anti-α-tubulin antibody (1D4) (Cat# T6199; Merk). Reactive bands were detected by Clarity Western ECL Substrate (Cat# 1705061 Bio-Rad). The intensity of bands was analyzed on a ChemiDoc station with Quantity-one software (Biorad, Milan, Italy).
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