hMADS cell lysates were obtained by using lysis buffer containing 50 mM Tris–HCl (pH 7.4), 1% NP-40, 1 mM EDTA, 150 mM NaCl, 1 mM sodium orthovanadate, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM phenylmethylsulfonylfluoride, and 50 mg/ml aprotinin. Samples were centrifuged, and protein concentrations were determined by the Bradford Protein Assay (Bio-Rad Laboratories, Segrate, Italy). Proteins were separated by SDS-PAGE then transferred to a nitrocellulose membrane using the Trans-Blot TurboTM Transfer system (Bio-Rad). To check loading and transfer efficiency, membranes were visualized with Ponceau S solution (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were then blocked for 1 h at room temperature (RT) in TBS-Tween-20 (50 mM Tris-HCL [pH 7.6], 200 mM NaCl and 0.1% Tween-20) containing 5% non-fat dried milk and subsequently incubated overnight at 4°C with the primary antibody (Table 1B ). After washing in TBS-Tween-20 and incubation with the secondary antibody for 1 h at RT (Table 1C ), bands were visualized with the Chemidoc Imaging system using the Clarity™ Western ECL chemiluminescent substrate (all from Bio-Rad). Quantitation of immunoreactive bands was performed using the Bio-Rad Image Lab software. Where appropriate, membranes were stripped, washed and re-probed for total protein content.
Ponceau s solution
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Ponceau S solution is a reversible protein stain used in Western blotting. It allows for the visualization of proteins on a nitrocellulose or PVDF membrane, facilitating the assessment of protein transfer efficiency and overall protein loading.
Automatically generated - may contain errors
Lab products found in correlation
Clarity ecl, by Bio-Rad (3 mentions)
Mini protean casting gels, by Bio-Rad (2 mentions)
Trans blot turbo, by Bio-Rad (2 mentions)
Chemidoc touch imaging system, by Bio-Rad (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Clarity western ecl chemiluminescent substrate, by Bio-Rad (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Ab116028, by Abcam (1 mentions)
Clarity max, by Bio-Rad (1 mentions)
Nupage transfer buffer, by Thermo Fisher Scientific (1 mentions)
0.2 m nitrocellulose membrane, by Bio-Rad (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Chemidoc touch system, by Bio-Rad (1 mentions)
Turboblot system, by Bio-Rad (1 mentions)
5 protocols using ponceau s solution
1
Protein Isolation and Western Blot Protocol
2
Optimized Western Blot Workflow
3
Phos-tag Western Blotting Protocol
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Yeast pellets were lysed and processed as described for Phos-tag Western blotting. The samples were loaded on home-made 6–14% acrylamide gradient Bis–Tris gels (0.33 M Bis–Tris pH 7.5) alongside broad molecular weight (10–245 kD) prestained protein ladder (ab116028; Abcam) and run at 130 V for 110 min using MES-SDS running buffer (Formedium MES-SDS5000). Gels were incubated in transfer buffer (NuPAGE Transfer Buffer with 20% ethanol final, NP0006; Thermo Fisher Scientific) and transferred onto 0.2-µm nitrocellulose membrane (1620112; Bio-Rad) at 25 V constant for 30 min using semidry transfer apparatus (1704150; Bio-Rad). Membranes were stained with Ponceau S solution (sc-301558; Santa Cruz) before imaging on a Bio-Rad ChemiDoc MP System to confirm equal protein loading. Membranes were then washed in Tris-buffered saline (TBS: 50 mM Tris–Cl, pH 7.5, 150 mM NaCl) and blocked in 5% nonfat dry milk in TBS. After further washes in TBS, membranes were incubated overnight at 4°C in primary antibody (diluted in 4% BSA, 0.02% sodium azide in TBS). After washing in TBS-T (TBS with 0.1% Tween-20), membranes were incubated in secondary antibody (diluted in TBS-T) for 1 h at room temperature, before washing again in TBS-T and developing using Clarity (1705061; Bio-Rad) or Clarity Max (1705062; Bio-Rad) ECL reagent on a Bio-Rad ChemiDoc MP System.
4
Western Blot Protocol for Protein Analysis
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Samples were run on homemade 6–14% Bis-Tris acrylamide gels. Two tubes of 6 ml mix were made up of 6% and 14% acrylamide, respectively (0.33 M pH 6.5 Bis-Tris, 0.083% APS and 0.083% TEMED). Then, 600 μl of the 14% solution was added to 2 × 1 mm Mini-Protean casting gels (Bio-Rad). The 14% solution was diluted with 600 μl of the 6% solution, and a further 600 μl was added to the casting gels. This was repeated until the gels were complete and a comb was added. After polymerization, extracts were loaded to a total of 25–50 µg protein per lane and run at 120 V for 2.5 h at 4 °C. Gels were then transferred onto 0.2 μm nitrocellulose membrane (Bio-Rad, 1620112) using a TransBlot Turbo (Bio-Rad) (30 min, 2.5 Amp). Membranes were stained with Ponceau S solution (Santa Cruz, sc-301558), imaged, cut, washed in TBS and blotted for at least 1 h with TBS containing 5% milk, washed three times in TBS-Tween and incubated with primary antibody overnight. Primary antibody was removed, and membranes were washed three times in TBS-T, incubated with secondary antibody for 1 h, washed three times in TBS-T Tween and imaged on a Chemidoc Touch imaging system (Bio-Rad) using Clarity ECL (Bio-Rad, 170-5061). Where indicated, blots were quantified by densitometry using FIJI with expression of the protein of interest normalized to Ponceau staining (loading control).
5
Bis-Tris Gel Electrophoresis and Western Blotting
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Samples (∼30 μg protein in each) were run on home-made 6–14% or 6–20% Bis-Tris acrylamide gels, poured as described6 (link) at 120V for 1.5–2 hours in MES-SDS running buffer (Formedium). Gels were incubated in transfer buffer (1x Nupage transfer buffer, NP0006-1, 20% Ethanol) and semi-dry blotting performed using blotting paper (Invitrogen, LC2008) and 0.2 μm nitrocellulose (BioRad 1620112) which had been pre-soaked in transfer buffer at 25V for 15 or 30 min on the BioRad TurboBlot system. Membranes were stained with ponceau S solution (Santa Cruz, sc-301558), imaged, then soaked in TBS-T (TBS +0.1% Tween 20) containing 5% milk powder for at least 1 hour, before washing and incubating with relevant primary antibodies (see key resources table ) overnight. Antibodies were then removed, membranes washed, and incubated with secondary antibodies (see key resources table ) for at least an hour before imaging. All membrane imaging was performed on a Chemidoc Touch system (BioRad) using Clarity ECL (BioRad).
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