SDS-PAGE was performed using NuPAGE 4-12% Bis-Tris gels (Life Technologies) according to the manufacturer's instructions. Gels were transferred on nitrocellulose membranes using an iBLOT (Life Technologies) according to the manufacturer’s instructions. Following Ponceau staining (Sigma), membranes were washed in TBS and blocked in TBS enriched with 5% milk powder or 3% PBS/BSA for 20 min at RT. Primary antibodies were incubated overnight at 4°C using a solution made of 1 μg/ml antibody in TBS supplemented with 1 mM CaCl2, 0.2% BSA and 0.02% Thymerosal. The membranes were then incubated using fluorescent antibodies (1/500 dilution in TBS supplemented with 1mM CaCl2 and 5% milk powder or 1% BSA), then revealed using a fluorescent scanner (Typhoon).
Ponceau staining
Manufactured by Merck Group
Sourced in United States, Italy
Ponceau staining is a reversible protein staining technique used in molecular biology and biochemistry to detect proteins on nitrocellulose or polyvinylidene difluoride (PVDF) membranes after electrophoretic transfer or dot blotting. It provides a quick and reliable way to visualize transferred proteins prior to immunodetection or other analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Immobilon p, by Merck Group (2 mentions)
Tween 20, by Merck Group (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Iblot, by Thermo Fisher Scientific (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Anti gst goat polyclonal, by GE Healthcare (1 mentions)
Genesnap, by Syngene (1 mentions)
Horseradish peroxidase hrp labeled secondary antibody, by Jackson ImmunoResearch (1 mentions)
Luminata forte western hrp substrate, by Merck Group (1 mentions)
Image studio lite, by LI COR (1 mentions)
Anti lamin b sc 6216, by Santa Cruz Biotechnology (1 mentions)
Trans blot nitrocellulose membrane, by Bio-Rad (1 mentions)
Clarity western ecl substrate kit, by Bio-Rad (1 mentions)
Tgx stain free fastcast acrylamide kit protocol, by Bio-Rad (1 mentions)
Trans blot turbo transfer system kit protocol, by Bio-Rad (1 mentions)
G box, by Syngene (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Pan akt, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
15 protocols using ponceau staining
1
Western Blot Protein Detection
2
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Thirty micrograms of total protein was separated by 15% SDS–PAGE. Proteins were then transferred to PVDF membranes (Immobilon-P, Millipore) and blocked with TBS-T (20 mM Tris, 137 mM NaCl, 0.1% Tween at pH 7.6) and 5% nonfat milk for 30 min at room temperature. After blocking, membranes were incubated with a specific primary antibody overnight at 4 °C. The following primary antibodies were used: 1:10000 rabbit monoclonal anti-thiophosphate ester (Epitomics, Inc., Burlingame, CA, USA), 1:1000 goat polyclonal anti-GST (GE Healthcare), 1:100 rabbit polyclonal anti-p27 (sc-528, Santa Cruz Biotechonology, Dallas, TX, USA) 1:1 000 rabbit polyclonal anti-PRC1 (sc-8356, Santa Cruz Biotechnology), 1:1000 anti-lamin B (sc-6216, Santa Cruz Biotechnology), 1:1000 anti-Na+/K+-ATPase (sc-58628, Santa Cruz Biotechnology) and 1:10,000 anti-GAPDH (CSB-PA00025A0Rb, Cusabio, San Diego, CA, USA). After several washes, membranes were incubated with horseradish peroxidase (HRP)-labeled secondary antibodies (Jackson Laboratories, West Grove, PA, USA) for 1 h at room temperature. After washing, bands were detected using Luminata Forte Western HRP Substrate (Millipore), and images were acquired using GeneSnap (Syngene) software. The density of the bands was analyzed using Image Studio Lite (Li-Cor) software, and Ponceau staining (Sigma-Aldrich) was used to normalize protein expression.
3
Western Blot Analysis of Calnexin and Galectin-3
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Each cell pellet was homogenized in 10× volume of RIPA lysis buffer (10 mM Tris-Cl pH 7.2, 150 mM NaCl, 1 mM EDTA pH 8) with 1% Triton X-10/0.1% deoxycholate, 0.1% SDS, and protease and phosphatase inhibitor mixture (Roche). Samples were then diluted in Laemmli's SDS sample buffer. Proteins were separated by electrophoresis on 10% polyacrylamide gels according to the TGX Stain-Free FastCast Acrylamide kit protocol (Bio-Rad), and transferred onto Trans-Blot nitrocellulose membranes (Bio-Rad) according to the Trans-Blot Turbo Transfer System kit protocol (Bio-Rad). Ponceau staining (Sigma-Aldrich) was performed to confirm that the samples were loaded equally. The membranes were blocked in 5% non-fat dry milk in TBS-T (pH 7.4, with 0.1% Tween-20) for 1 h at room temperature. Primary antibodies were diluted in 3% BSA (Sigma-Aldrich) in TBS-T [mouse anti-calnexin 1:3,000 (Genetex) rat anti-mouse/human Gal-3 (E-Bioscience; 1:1,000)], and the membranes were incubated overnight at 4°C. The primary antibody was removed, and the blots were washed in TBS-T and then incubated for 45 min at room temperature in HRP-conjugated secondary antibodies [anti-mouse (Biorad) anti-rat (Amersham)]. Reactive proteins were visualized using a Clarity Western ECL substrate kit (Bio-Rad), and exposure was performed using UVItec (Cambridge MINI HD). Images were acquired by NineAlliance software.
4
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After the respective treatments, animals were anesthetized. Samples from the hypothalamus and soleus muscle were obtained, minced coarsely, and homogenized immediately in solubilization buffer containing 100 mM tris (pH 7.6), 1% Triton X-100, 10 mM Na3VO4, 100 mM NaF, 10 mM Na4P2O7, 4 mM EDTA, 150 mM NaCl, aprotinin (0.1 mg/ml), and phenylmethylsulfonyl fluoride (35 mg/ml) using a Polytron PTA 20S generator (model PT 10/35, Brinkmann Instruments, Westbury, NY) operated at maximum speed for 30 s and clarified by centrifugation. All the samples from all groups were subjected to SDS-PAGE and blotted onto a nitrocellulose membrane. The membranes were incubated for 12 hours at 4°C with primary antibody after blockade with 5% nonfat milk in tris-buffered saline with Tween 20 (TBST) (10 mM tris, 150 mM NaCl, and 0.02% Tween 20) for 90 min at room temperature. After the secondary antibody incubation, the signal was detected for 60 min in a 3% nonfat milk–TBST solution, treated with 2 ml of SuperSignal West Pico Chemiluminescent Substrate, exposed to photosensitive RX film from Kodak, or visualized using G:Box from SynGene. Band intensities were quantified by optical densitometry using UN-SCAN-IT gel 7.1 (Silk Scientific Inc.). Ponceau staining from Sigma-Aldrich (St. Louis, MO, USA) was also used to monitor the loading control for each sample.
5
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells cultured in 6-well plates were lysed using Laemmli buffer. Lysates were homogenized with TissueLyser (Qiagen, Milano, Italy) and heated to 99 °C. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare Life Sciences, Chicago, IL, USA). Sample loading and transfer was checked via Ponceau staining (Sigma-Aldrich, St. Louis, MO, USA). Membranes were blocked using 5% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) in TBS with 0.1% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA). Membranes were incubated with antibodies against HDAC3 (Santa Cruz Biotechnology, Dallas, TX, USA), phospho-AKT Ser473 and pan-AKT (Cell Signaling Technology, Danvers, MA, USA), antibody cocktail to detect mitochondrial proteins (Total OXPHOS Rodent WB Antibody Cocktail, Abcam, Cambridge, UK), PPARγ (Cell Signaling Technology, Danvers, MA, USA) or HSP90 antibody (Santa Cruz Biotechnology, Dallas, TX, USA) as a loading control. HRP-conjugated secondary antibodies were used for detection with chemiluminescence (Pierce ECL Western Blotting Substrate, Life Technologies Italia, Monza, Italy). Protein levels were quantified and normalized to HSP90 using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
6
Western Blot Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoblot analysis was performed as described previously [8 (link)]. In short, all lysates for immunoblot analysis were prepared by lysing cells in PBS‐1%TritonX‐100 buffer supplemented with Protease Inhibitor Cocktail and Phosphatase inhibitor (Roche, Basel, Switzerland). After that, the resulting suspension was incubated on ice for 10 min, centrifuged, and the supernatant collected. Tissue lysates were made in the mentioned lysis buffer using Precellys 24 tissue homogenizer (Bertin instruments) and the Precellys kit (#KT03961‐1‐003.2). Proteins were separated on gradient SDS gels (BioRad) and transferred to nitrocellulose membranes (BioRad #1704158). Transfer quality was evaluated with Ponceau staining (Sigma, #P7170). Immunoblots were quantitated using image j (Bethesda, MD, USA). Statistical significance was determined via a one‐sided t‐test, unless specified otherwise in the figure legends.
7
Protein Expression Analysis by SDS-PAGE
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein analysis was performed by SDS-PAGE electrophoresis on 12% polyacrylamide tris-glycine, protein transfer to a nitrocellulose membrane (Amersham, GE Healthcare Europe GmbH, Milano, Italy), revelation by Ponceau staining (Sigma Chemical Co., Milano, Italy), and immunoblotting with specific antibodies. Electrophoresis separation and western blotting were carried out on equal amounts (as protein) of homogenate or MEF samples, in order to investigate protein level expression in cellular membranes. Nitrocellulose membranes were blocked in TBS-Tween 0.1% or 0.2% buffer containing 5% non-fat milk or 3–5% BSA and probed with specific antibodies diluted in the same solution. Immunoblottings were performed using anti-cPLA2 (1:200), anti-COX2 (1:1000), anti-LC3 I-II (1:1000), anti-p62 (1:1000), anti Beclin-1 (1:1000), and anti-β-actin (1:1500) antibodies. Immunoreactive proteins were revealed by ECL and semi-quantitatively estimated by LAS4000 Image Station (GE Healthcare Italia, Milano, MI, Italy). Normalization in the same sample was carried out with respect to β-actin homogenate samples [44 (link),45 (link)] and to the total amount of proteins detected by Ponceau staining in MEFs, permitting a straightforward correction for lane-to-lane variation [46 (link),47 (link)].
8
Western Blot Analysis of Extracellular Vesicles
9
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from a part of the ground GAS muscles. RIPA Lysis Buffer (Sigma, Poole, UK) containing Pierce Protease and Phosphatase Inhibitor Mini Tablets, Ethylenediaminetetraacetic acid (EDTA)-free (Thermo Fisher Scientific, Waltham, MA, USA). The samples were homogenized and sonicated for 30 s twice, and then centrifuged for 10 min at 14,000×g at 4 °C. Supernatant was collected and total protein content determined with a BCA Protein Assay (Sigma, Poole, UK). 30 or 45 μg protein extract were electrophoresed and separated on NuPAGE™ 4–12 %, Bis-Tris, 1.0–1.5 mm, Mini Protein Gels (Invitrogen, Renfrewshire, UK) and transferred to a polyvinylidene fluoride (PVDF) membrane. GAPDH (ab8245, 1:2000) or ponceau staining (Sigma, Poole, UK) were used as loading control. Membranes were blocked with 5 % bovine serum albumin (BSA) (Sigma, Poole, UK) in tris-buffered saline (TBS) for an hour and were cut to probe with primary antibodies which were optimised prior to experiments. Pre-cut blots were used for the antibody treatment overnight. After incubating with a fluorescence secondary antibody, blots were imaged on a the Licory Odissey CLx (Licor, Bad Homburg, Germany) and band densities were analysed using ImageJ. Results were normalized to the loading control.
10
Immunoblot Analysis of Purified Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunoblot analyses of purified proteins or cell lysates, proteins were separated on SDS-PAGE (with or without DTT as indicated in the figures) and subsequently blotted to nitrocellulose membrane (Invitrogen) with Bio-Rad Trans-Blot® Turbo™ (25 V, 7 min). Equal loading of cell lysate proteins (30 μg per lane) was confirmed using Ponceau staining (Sigma), while chromatography fractions were instead loaded with equal volumes in each lane. Blocking was done with 5% powdered milk (Carl Roth) and 0.5% BSA (Bovine Serum Albumin, Sigma-Aldrich) in TBS-T (Tris Buffered Saline (Bio-Rad) with 0.5% Tween 20, Sigma-Aldrich). Primary antibodies were obtained from either Santa Cruz (SC) or Cell Signaling Technology (CST): anti-Trx1 (SC catalog number: 166393; 1:500), anti-TXNL1 (SC: 515218; 1:1000), anti-TrxR1 (CST#6925, 1:1000), anti-HSP27 (CST#2404, 1:1000), anti-insulin B chain (SC: 377071; 1:1000) and anti-β-actin (SC: 47778, 1:1000). The membranes were incubated overnight with the primary antibodies (diluted in TBS-T), then after washing, the corresponding HRP-conjugated secondary antibodies (Dako, P-0447, 1:8000 or Dako, P-0399, 1:3000) were added to the membranes for 1 h at RT (diluted with 5% powdered milk and 0.5% BSA in TBS-T). After addition of ECL substrate (BR1705062), chemiluminescent signals were detected using a Bio-Rad ChemiDoc XRS scanner.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!