After removing spent media and washing cells with cold phosphate buffered saline (PBS), the cells were incubated with cold Pierce RIPA lysis buffer (Thermo Scientific, Hudson, NH) containing Halt™ Protease Inhibitor Single-Use Cocktail, Halt™ Phosphatase Inhibitor Single-Use Cocktail (Thermo Scientific) and 1 mM dithiothreitol (DTT) for 15 min with occasional swirling. Then, the cells were scraped, homogenized with a 26-gauge needle and vortexed at the highest setting for 1 min; the lysates were cleared by centrifuging at 16,000 g at 4°C for 15 min. Protein concentration was determined with the bicinchoninic acid (BCA) method (BCA Protein Assay - Reducing Agent Compatible; Thermo Scientific). Proteins were separated on NuPAGE 4–12% Bis Tris gel electrophoresis (Life Technologies), and transferred to a nitrocellulose membrane (iBlot Gel Transfer Stacks Nitrocellulose; Life Technologies) using iBlot Gel Transfer Device (Life Technologies). The membrane was probed with monoclonal rabbit antibodies, anti-HMGCR (1:500, ab174830, Abcam Inc., Cambridge, MA), anti-vimentin (1:1000, ab92547, Abcam), and anti-E-cadherin (1:1000, 24E10, Cell Signaling Technology, Beverly, MA). A monoclonal mouse antibody to β-actin (1:500, ab8226, Abcam) was used as a loading control. Immunodetection was performed using the iBlot Western Detection chromogenic kit (Life Technologies).
Iblot gel transfer stacks nitrocellulose
Manufactured by Thermo Fisher Scientific
Sourced in United States
The IBlot Gel Transfer Stacks Nitrocellulose is a lab equipment product designed for the transfer of proteins from polyacrylamide gels to nitrocellulose membranes. It is a component used in the Western blotting technique.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab174830, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Pierce ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Halt protease inhibitor single use cocktail, by Thermo Fisher Scientific (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Nupage 4 12 bis tris gel electrophoresis, by Thermo Fisher Scientific (1 mentions)
Bca protein assay reducing agent compatible, by Thermo Fisher Scientific (1 mentions)
Iblot western detection chromogenic kit, by Thermo Fisher Scientific (1 mentions)
Halt phosphatase inhibitor single use cocktail, by Thermo Fisher Scientific (1 mentions)
Iblot gel transfer device, by Thermo Fisher Scientific (1 mentions)
Ab92547, by Abcam (1 mentions)
Western blotting luminol reagent, by Santa Cruz Biotechnology (1 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (1 mentions)
Nupage novex bis tris 4 12 midi gels, by Thermo Fisher Scientific (1 mentions)
Am4300, by Cell Signaling Technology (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
8 protocols using iblot gel transfer stacks nitrocellulose
1
Analyzing Protein Expression in Cell Lysates
2
Western Blotting of GAPDH and LC3B
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Western blotting was performed as described previously [14 (link)]. Briefly, equal amounts of proteins across conditions were separated on NuPAGE Novex Bis-Tris 4–12% Midi gels (Life Technologies) at room temperature and constant voltage. Proteins were transferred to a nitrocellulose membrane (iBlot Gel Transfer Stacks Nitrocellulose) using an iBlot dry blotting system (Life Technologies). After blocking, the membranes were incubated overnight at 4 °C with primary antibodies: mouse monoclonal antibody against glyceraldehyde phosphate dehydrogenase (GAPDH; 1:5000; Thermo Fisher Scientific AM4300, RRID: AB_437392), or rabbit polyclonal antibody against LC3B (1:100; Cell Signaling Technology #2775, RRID: AB_915950, although this antibody was directed against LC3B, cross-reactivity may exist with other LC3 isoforms according to the manufacturer). After extensive washing, the membranes were incubated with peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG antibodies (1:5000; Jackson ImmunoResearch, Cambridgeshire, United Kingdom) and developed with a chemiluminescent substrate (Western Blotting Luminol Reagent; Santa Cruz Biotechnology, Dallas, TX, USA) in a MicroChemi imaging system (Bioimaging Systems, Upland, CA, USA). Uncropped blots are shown in Figure S1 . GAPDH was used as a loading control.
3
Protein Extraction and Western Blot Analysis
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Total cell extracts were lysed in “NP40 buffer” (50 mM Tris-HCl , 400 mM NaCl, 5 mM EDTA , 1% IGEPAL , 0.2% SDS) complemented with protease and phosphatase inhibitors (11836145001 and 04906837001, Roche) and then incubated on ice for 30 min. Extracts were then sonicated for 10 min (Diagenode Bioruptor, 10 cycles, 30′′ on / 30′′ off). Protein concentrations from total cell extracts were determined using a Pierce BCA Protein Assay kit (Thermo Scientific). Total cell extracts were run on 4%–12% Bis-Tris gels (Invitrogen) and transferred on nitrocellulose membranes (iBlot Gel Transfer Stacks Nitrocellulose, Invitrogen). Membranes were washed in TBST (20 mM Tris, pH 7.6 , 130 mM NaCl , 0.1% Tween 20) and blocked in 5% (w/v) dry nonfat milk or 5% (w/v) bovine serum albumin (Sigma) for primary phospho-antibodies. Membranes were then incubated with primary antibodies (overnight, 4°C) and washed before being incubated with secondary HRP-conjugated antibodies for 1 h. Primary and secondary antibodies are listed in Supplemental Table S1 . Image acquisitions were performed using the ChemiDoc Touch Imaging System (Bio-Rad), and quantification was performed using Image Lab software (v.5.2.1, Biorad) and normalized with histone H3 or total nonphosphoprotein. Statistical analyses on means were made using Student's t-tests (unilateral, paired, P < 0.05).
4
Western Blot Analysis of ERG, PTEN, and β-Actin
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Cells were lysed in modified RIPA buffer (Santa Cruz) supplemented with PMSF, protease inhibitor and sodium orthovanadate. Protein concentration of the lysates were determined using BCA protein assay reagent (Bio-Rad); 30μg of the extracted protein was mixed with Laemmli sample buffer containing β-ME, denatured, separated using 10% PAGEr Gold Precast Gels (Lonza), and transferred using iBlot Gel Transfer system (Invitrogen) and iBlot Gel Transfer Stacks Nitrocellulose (Invitrogen). Anti-ERG (#2805-1, Epitomics), anti-PTEN (#S-0271, Epitomics), anti-β-actin antibody (Santa Cruz Biotech), antibodies were used at 1:1000 dilution with blocking with 5% skim milk. After incubation with primary antibodies overnight at 4°C, horseradish peroxidase–labeled secondary antibodies were then applied to the membranes for 1 h at room temperature. Signals were visualized using ECL Western blotting detection substrate (Thermo Scientific).
5
Nuclear and Cytoplasmic Protein Fractionation of i-Neurons
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Nuclear and cytoplasmic protein fractions were extracted and separated from 100 day i-neurons using NE-PERTM Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher, Scientific, Waltham, MA, USA) supplemented with protease and phosphatase inhibitors (Roche) following the manufacturer’s instructions. Protein concentration was determined using DC Protein Assay (Biorad, Hercules, CA, USA). Nuclear extracts (15 μg) were separated on a 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose (iBlot Gel Transfer Stacks Nitrocellulose, Thermo Fisher, Scientific Waltham, MA, USA). After blocking with 5% non-fat dry milk, TS1X Buffer (20mM Tris-HCl pH 7.5, 150 mM NaCl) and 0.3% Tween-20 for 1 h, membranes were incubated with primary antibodies (Anti-MeCP2 C-ter (1:500) ab2829, Abcam; Anti-MeCP2 N-ter (1:500) M7443, Sigma-Aldrich; Anti-Histone H3 (1:2000) ab1791, Abcam) overnight at 4 °C, and then with secondary antibodies (goat Anti-Mouse HRP AP124P (1:3000), goat Anti-rabbit HRP AP307P (1:3000), both from Millipore) for 2 h at RT. The ClarityTM Western ECL Substrate (Biorad, Hercules, CA, USA) was used for detection of the HRP-conjugated secondary antibody.
6
Muscle Protein Extraction and Western Blot Analysis
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Total proteins were isolated from 50 mg of whole hind limb muscles using T‐PER tissue protein extraction reagents (78510; Thermo Fisher Scientific, Inc.). Protein concentration was measured using the BCA protein assay kit (Pierce 23225; Thermo Fisher Scientific, Inc.). Total proteins (20 μg) were separated on Bolt 4–12% Bis‐Tris PlusGels (NW04125BOX; Thermo Fisher Scientific, Inc.) by electrophoresis, and the fractionated proteins were subsequently transferred to a nitrocellulose membrane using iBlot Gel Transfer Stacks Nitrocellulose (IB23001; Thermo Fisher Scientific, Inc.). The blots were probed using the following antibodies: AMP‐activated protein kinase α (AMPKα; SC‐74461; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), Thr172‐phosphorylated (p)‐AMPKα (sc‐33524, Santa Cruz Biotechnology, Inc.), AKT (no. 4691; Cell Signaling Technology, Inc., Danvers, MA, USA), Ser473‐p‐AKT (#4060; Cell Signaling Technology, Inc.), glucose transporter 4 (GLUT4; ab64; Abcam, cambridge, England) and β‐actin (sc‐477778; Santa Cruz Biotechnology, Inc.). The density of protein bands was analyzed using Bio‐Rad imaging software (Bio‐Rad Laboratories, Hercules, CA, USA). The individual values were originally expressed as a ratio of a standard (β‐actin content) and then expressed as a fold change of the control group value.
7
Western Blot Analysis of Shiga Toxin
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To detect the Stx subunits, a Western Blot analysis was performed. SDS-Gels were prepared as described above and blotted using an iBlot 2 Gel Transfer Device (Life Technologies) and Nitrocellulose iBlot Gel Transfer Stacks (Invitrogen). Blotting was performed for 10 min. 0.1% Tris Buffered Saline (TBS, composed of Tris (PanReac) and NaCl (PanReac) and puffered to a pH of 7.5 with HCl)-Tween solution (TBS-T, Tween-20 Applichem) was used for washing membranes three times. Subsequently, membranes were blocked using 2% (w/v) BSA (Sigma Aldrich) TBS-T solution over night at 4 °C. A primary mouse monoclonal antibody was tested, namely the Shiga toxin 2 Antibody 11E10 (sc-52727, Santa Cruz). The antibody was diluted 1:1000 in 1% (w/v) BSA TBS-T solution and the membranes were incubated for 2 h at room temperature (RT). After washing the membranes three times with TBS-T, the secondary antibody Horse Radish Peroxidase (HRP)-conjugated goat anti-mouse IgG (Azure Biosystems) 1:3000 in 1% (w/v) BSA TBS-T solution. Membranes were incubated with the secondary antibody for 1.5 h at RT. The HRP coupled to the secondary antibody, was used for detection by WesternBright ECL HRP (Biozym). Illumination signal was detected using a AzureWestern C600 Imaging System (Azure Biosystems).
8
Western Blot Analysis of Signaling Proteins
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Cells were lysed in a buffer containing 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS and 50 mM Tris, pH 8.0 for 1 h. The supernatant was collected after centrifugation, and 6 μg of protein was used for Western blotting. Briefly, the proteins were separated on NuPage 4–12% Bis-Tris gels (Invitrogen) by electrophoresis and transferred to nitrocellulose i-Blot gel transfer stacks (Invitrogen). The membrane was washed with PBS with 0.1% Tween 20 (PBST), placed in blocking buffer (PBST containing 5% nonfat milk) for 1 h. Then the membrane was incubated with primary antibodies at 4°C overnight. The primary antibodies used were rabbit anti-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (IκB-α) (Cell Signaling Technology, Danvers, MA; 1:1,000), phosphorylation interferon regulatory factor 3 (pIRF3) (Cell Signaling Technology; 1:1,000), and mouse monoclonal anti-β-actin (Sigma; 1:5,000) as an internal control. After washing with PBST, membranes were incubated with a horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (Cell Signaling Technology; 1:5,000) or goat anti-rabbit IgG (Cell Signaling Technology; 1:1,000) as secondary antibodies. Specific proteins were visualized using Immunostar LD reagent (Wako Pure Chemical, Osaka, Japan), and the chemiluminescence was detected using the C-DiGit blot scanner (LI-COR).
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