Proteins were extracted from cells using Pierce RIPA Buffer (Thermo Scientific) titrated to 1% SDS (Bio-Rad) with the addition of Halt Protease Inhibitor Cocktail (Thermo Scientific) and Benzonase Nuclease (Sigma). The protein concentration was determined using Pierce Rapid Gold BCA Protein Assay (Thermo Scientific) according to the manufacturer’s protocol. Total protein (35ug) was loaded for electrophoresis into 4–20% sodium dodecyl sulfate polyacrylamide gels (Bio-Rad). Separated proteins were transferred to a 0.22 µm nitrocellulose membrane (Bio-Rad). Membranes were blocked with Odyssey Blocking Buffer (Li-Cor) diluted 1:1 with tris buffered saline with 0.1% Tween 20 (TBS-T). Membranes were then cut into different strips and stained with antibodies against GAPDH (D16H11 Cell Signaling Technology #5174, 1:1000 dilution), p53 (7F5, Cell Signaling Technology #2527, 1:1000 dilution), PHLDA3 (Cell Signaling Technology #4294, 1:1000 dilution), and p21 Waf1/Cip1 (12D1 Cell Signaling Technology #2947, 1:1000 dilution) followed by secondary fluorescently-conjugated IRDye 800CW (Li-Cor, 1:20,000 dilution). Bands were visualized at 700 and 800 nm using the Odyssey CLX imaging system (Li-Cor).
0.22 m nitrocellulose membrane
Manufactured by Bio-Rad
Sourced in United States
The 0.22 µm nitrocellulose membrane is a laboratory filtration product used for the separation and purification of biomolecules. It has a pore size of 0.22 micrometers, which allows for the effective retention of small particles and molecules while allowing the passage of liquids and smaller dissolved species.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (2 mentions)
Pierce ripa buffer, by Thermo Fisher Scientific (1 mentions)
Benzonase nuclease, by Merck Group (1 mentions)
Sodium dodecyl sulfate polyacrylamide gel, by Bio-Rad (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Irdye 800cw, by LI COR (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Pierce rapid gold bca protein assay, by Thermo Fisher Scientific (1 mentions)
Immobilon western, by Merck Group (1 mentions)
Anti β catenin, by Santa Cruz Biotechnology (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Anti tcf4, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg, by Agilent Technologies (1 mentions)
Gapdh, by Bio-Techne (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Amersham imagequant, by Cytiva (1 mentions)
Anti ha, by Cell Signaling Technology (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
7 protocols using 0.22 m nitrocellulose membrane
1
Western Blot Analysis of Protein Targets
2
Western Blot Analysis of Transfected C33A Cells
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C33A cells were cultured in 60 mm dishes and transfected with 3 µg of the indicated plasmid. 48 h post-transfection, cells were lysed using 300 µL of RIPA buffer (100 mM Tris pH 8.0, 50 mM NaCl2, 0.5% Nonidet P-40, and protease inhibitor cocktail (Roche, Basel, Switzerland)). 20 μg of cell protein extracts were analyzed by SDS-PAGE gels (10–12%) and transferred in a 0.22 µm nitrocellulose membrane (Bio-Rad). Membranes were blocked with 10% skimmed milk in TBS-0.1% Tween 20 per 1 h at room temperature, followed by incubation with the indicated primary antibody diluted 1:1000: anti-FLAG M2 (Sigma Aldrich, Sant Louis, MO, USA); anti-TCF-4 (Santa Cruz Biotechnologies, Dallas, TX, USA); anti-β-catenin (Santa Cruz Biotechnologies, Dallas, TX, USA). After washing three times with TBS-0.1% Tween 20, membranes were incubated with HPR-conjugated secondary anti-mouse antibody in a dilution 1:10000 (Santa Cruz, Biotechnologies, Dallas, TX, USA). Proteins were visualized utilizing the Immobilon Western (Millipore) according to the manufacturer’s instructions. Western blots were performed at least three times each to assure result reproducibility.
3
Western Blot Analysis of Protein Lysates
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Total protein lysates were isolated in a RIPA buffer (150 mM NaCl, 1 mM EDTA, 50 mM Tris/HCl, 0.5% SDS, 1% NP-40), plus a HaltTM protease and phosphatase inhibitor cocktail (Thermo Fisher, cat #78442). Protein was quantified by a BCA protein assay kit (Thermo Fisher; cat. #PI23227). 50–100 μg protein lysate was separated on polyacrylamide gels and transferred to a 0.22 µM nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Membranes were immunoblotted with primary antibodies for NOVA1 (MilliporeSigma, Burlington, MA, USA; cat. #07-637), NOVA2 (Proteintech, Rosemont, IL, USA; cat. #55002-1-AP), BIM (Cell Signaling Technology Inc., Danvers, MA, USA; cat. #2933, clone C34C5), or GAPDH (Trevigen, Wiesbaden, Germany; cat. #2275-PC-100) in a milk blocking buffer. Proteins were detected using goat anti-rabbit IgG (Dako Agilent, Santa Clara, CA, USA; cat. #P0448) secondary antibody labelled with HRP followed by signal visualization western blot imaging system (Amersham ImageQuant 800 western blot imaging system, Cytiva Life Science, Marlborough, MA, USA).
4
Labeling Murine Red Blood Cells
5
Western Blot Analysis of HA and H4 Proteins
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C33-EV, C33-E616, and C33-E716 cells were cultured in 60 mm dishes and after 24 h lysed using 300 µL of RIPA buffer (100 mM Tris pH 8.0, 50 mM NaCl2, 0.5% Nonidet P-40, and protease inhibitor cocktail (Roche, Basel, CH)). A total of 20 μg of cell protein extracts were analyzed by SDS-PAGE gels (10–12%) and blotted onto a 0.22 µm nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 10% skimmed milk in TBS-0.1% Tween 20 for 1 h at room temperature, followed by incubation with anti-HA (Cell Signaling, Danvers, MA, USA) and anti-H4 (Cell Signaling, Danvers, MA, USA) primary antibodies diluted 1:1000 and 1:20,000, respectively. After washing three times with TBS-0.1% Tween 20, membranes were incubated with HRP-conjugated secondary anti-mouse antibody (Santa Cruz, Bio., Dallas, TX, USA) in a dilution 1:10,000. Proteins were visualized utilizing the Clarity™ Western ECL Substrate (Bio-Rad, Hercules, CA, USA), according to the manufacturer’s instructions. Then, membranes were visualized and analyzed in the iBright FL1500 imagining system (Invitrogen, Waltham, MA, USA).
6
Botrocetin Purification and Analysis
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Ponceau S (code P3504), glycerol (code G9012), Nα-Benzoyl-DL-arginine 4-nitroanilide hydrochloride (code B4875), dimethylsulfoxide (DMSO, code 472301), gelatin from porcine skin (code G8150), 1-naphthalamine (code N9005), peroxidase-conjugated anti-horse IgG (code A6917), and sodium nitrite (code 237213) were obtained from Sigma (USA). Bis-acrylamide was purchased from Ludwig Biotec. Tricine (code 22561) was obtained from USB corporation (USA), and agarose (type VII, code V3125) and acrylamide (code V3115) were from Promega. Polyclonal anti-botrocetin antibodies were raised in rabbits [61 (link)], and commercial Bothrops antivenin was a kind donation of Institute Butantan, São Paulo, SP, Brazil (lot 0611203). AlexaFluor 647-conjugated anti-rabbit IgG (code A21245) was purchased from Invitrogen (USA). Coomassie brilliant blue R-250 (code 161-0400), Coomassie brilliant blue G-250 (code 161-0406), the molecular mass standard (code 61-0376), and 0.22-µm nitrocellulose membranes (162-0112) were obtained from Bio-Rad. HD-Phe-Pip-Arg-pNA (code S-2238) was purchased from Chromogenix (USA). Triton X-100 (code 11869), imidazole (code 4716), and urea (code 57136) were obtained from Merck. Tris-base (code 1027) and glacial acetic acid (code 1019) were obtained from Synth.
7
Western Blot Analysis of Bacterial Samples
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Bacterial whole cell preps (in TPIG = 50 mM Tris 8.0, protease inhibitor cocktail [Sigma-Aldrich, St. Louis, MO, USA. P2714], 10% glycerol) were boiled in Laemmli sample buffer for 10 min. and were loaded at 108 cells per lane onto NuPAGE 4–12% Bis-Tris Protein Gels (Invitrogen). The NuPAGE MES SDS running buffer was used and gels were run variously at 50–120 V. Resolved gels were transferred to 0.22 µm nitrocellulose membranes (BioRad, Hercules, CA, USA). Membranes were blocked for 30 min. in 5% non-fat dry milk in PBS 0.05% Tween 20 (PBST); except when anti-goat antibodies were used, in which case membranes were blocked for 30 min. with 5% heat-inactivated FBS in PBST. Primary antibodies were used at the indicated concentrations for an overnight incubation at 4 °C in PBST. Secondary horseradish peroxidase (HRP)-conjugated antibodies were diluted to the indicated concentrations in PBST and incubated at 4 °C for 1 h. Where indicated, a biotinylated secondary antibody was used, followed by streptavidin-linked HRP, each for 1 h incubations at 4 °C. The development of chemiluminescent substrate (SuperSignal West Pico or SuperSignal West Femto Maximum Sensitivity, Pierce) was visualized using the BioRad ChemiDoc Touch Imaging System. Densitometry was analyzed using the BioRad Image Lab 6.0 software.
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