Myocytes were cultured in D-MEM with Sodium Pyruvate, without Amino Acids (WAKO, Osaka, Japan) for 1 h to lower mTORC1 activity. The cells were then cultured with DMEM containing amino acids and 100 nM insulin (Nacalai Tesque) for 10 min. After washing with cold PBS, the cells were lysed in RIPA buffer containing 1% (v/v) PIC and thoroughly sonicated on ice. Proteins were isolated from the lysate as described above. The isolated proteins (20 µg) were separated by electrophoresis on NuPAGE Novex 3–8% Tris-Acetate Protein Gel (Thermo Fisher Scientific) at 150 V for 60 min for S6K and pS6K, or on Extra PAGE One Precast Gel 15% (Nacalai Tesque) at 300 V for 30 min for 4E-BP1, p4E-BP1 and LC3, and transferred to a nitrocellulose membrane using an iBlot system (Thermo Fisher Scientific) with the program, P0, 9 min. The membrane was blocked with PBS-T containing 1% (w/w) skim milk and then incubated with primary antibody solution at 4 °C overnight. After washing with PBS-T three times, the membrane was incubated with secondary antibody solution for 1 h at room temperature. The blots were developed by Pierce Western Blotting Substrate Plus (Thermo Fisher Scientific) or ImmunoStar LD (WAKO). The bands were digitally detected by ChemiDoc XRS+ (Bio-Rad, Hercules, CA, USA) and quantified by Quantity One software (Bio-Rad).
Extra page one precast gel
Manufactured by Nacalai Tesque
Sourced in Japan
Extra PAGE One Precast Gel is a laboratory product designed for gel electrophoresis applications. It provides a pre-cast polyacrylamide gel for the separation and analysis of protein or nucleic acid samples.
Automatically generated - may contain errors
Lab products found in correlation
Cbb stain one super, by Nacalai Tesque (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Las4000, by GE Healthcare (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Pierce western blotting substrate plus, by Thermo Fisher Scientific (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Iblot system, by Thermo Fisher Scientific (1 mentions)
Nupage novex 3 8 tris acetate protein gel, by Thermo Fisher Scientific (1 mentions)
Insulin, by Nacalai Tesque (1 mentions)
Sample buffer solution with 2 me, by Nacalai Tesque (1 mentions)
Horseradish peroxidase conjugated anti rabbit and anti mouse igg antibodies, by GE Healthcare (1 mentions)
Hikari solution a, by Nacalai Tesque (1 mentions)
Chemi lumi one ultra, by Nacalai Tesque (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Ecl plus western blotting detection system, by GE Healthcare (1 mentions)
Pierce ecl plus western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Nacalai Tesque (1 mentions)
Pd l1, by Cusabio (1 mentions)
Goat anti mouse igg hrp conjugate, by Merck Group (1 mentions)
10 protocols using extra page one precast gel
1
Western Blot Analysis of mTORC1 Pathway
2
SDS-PAGE Analysis of ICG-Antibody Conjugates
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ICG–antibody conjugates were run on a 5–20% polyacrylamide gel (Extra PAGE one Precast gel, Nacalai Tesque) in Tris-glycine-SDS buffer, 200 V for 40 min. NIR fluorescence (Ex: 760 nm/Em: 830 nm) of ICG was detected first, and then proteins were stained with Coomassie Brilliant Blue (CBB Stain One Super, Nacalai Tesque). Antibody molecules were reduced by dithiothreitol (DTT).
3
Western Blot Analysis of Muscle Proteins
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Cells were harvested and disrupted in a Sample Buffer Solution with 2-ME (Nacalai Tesque Inc.). Samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis using a 5–20% Extra PAGE One Precast Gel (Nacalai Tesque Inc.) and electrotransferred to a polyvinylidene fluoride membrane with the Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Hercules, CA; 2.5 A, 25 V, 7 min). The membrane was blocked for 1 h in 5% (w/v) skimmed milk in Tris-buffered saline containing 0.05% (v/v) Tween 20, then incubated with primary antibodies in Hikari Solution A (Nacalai Tesque Inc.), followed by incubation with secondary antibodies and detection using Chemi-Lumi One Ultra (Nacalai Tesque Inc.).
The following primary antibodies were used for western blotting: rat anti-MyoD (5F11, Millipore 1 : 500), anti-Mettl3 (15073-1-AP, Proteintech, 1 : 1000), anti-Hsp90 (H-114, Santa Cruz Biotechnology, 1 : 1000) and mouse anti-HuR (3A2, Santa Cruz Biotechnology, 1 : 200). Secondary antibodies were horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG antibodies (GE Healthcare, 1 : 5000).
The following primary antibodies were used for western blotting: rat anti-MyoD (5F11, Millipore 1 : 500), anti-Mettl3 (15073-1-AP, Proteintech, 1 : 1000), anti-Hsp90 (H-114, Santa Cruz Biotechnology, 1 : 1000) and mouse anti-HuR (3A2, Santa Cruz Biotechnology, 1 : 200). Secondary antibodies were horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG antibodies (GE Healthcare, 1 : 5000).
4
Protein Extraction and Western Blot Analysis of iPSC-CMs
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The total protein of iPSC-CMs was extracted using radioimmunoprecipitation assay (RIPA) buffer containing a phosphatase inhibitor and protease inhibitor for 30 min on ice. After centrifugation at 20,000g for 20 min, the supernatant was used as total protein lysate. The samples were separated via electrophoresis on 5–20% Extra PAGE One precast gel (Nacalai Tesque). The fractionated samples were transferred to a nitrocellulose membrane. The membrane was blocked using 5% BSA in Tris-buffered saline with Tween 20 (TBS-T) for 1 h at room temperature, followed by reaction with primary antibodies for 24 h at 4 °C. Next, the membranes were reacted with secondary antibody for 1 h at room temperature, followed by treatment with Pierce ECL Plus western blotting substrate (Thermo Fisher Scientific) for 5 min. The chemiluminescent signals were detected using the ECL Plus western blotting detection system (GE Healthcare Life Sciences) and the images were captured using ImageQuant LAS4000 (GE Healthcare Life Sciences). The primary and secondary antibodies used are shown in Supplemental Table 3.
5
Western Blot Analysis of Cancer Markers
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The cells were lysed with RIPA buffer (Nacalai Tesque) for 15 min on ice and centrifuged at 10 000×g for 10 min at 4 °C to remove insoluble material. Protein concentration was determined by a Protein Assay CBB Solution (Nacalai Tesque). The cell lysates (24 μg) were separated by electrophoresis on 5–20% Extra PAGE One Precast Gel (Nacalai Tesque) at 200 V for 45 min. The protein was transferred to PVDF membrane by a Trans-Blot Turbo Transfer System (BIO-RAD). The membranes were incubated in 5% skim milk for 1 h at room temperature to prevent nonspecific binding. Then, the membranes were incubated in primary antibodies against HER2 (1 : 5000, Santa Cruz Biotechnology), EGFR (1 : 100, Santa Cruz Biotechnology), VEGFR-2 (1 : 200, Santa Cruz Biotechnology), PD-L1 (1 : 500, CUSABIO) or beta-actin (1 : 200, Santa Cruz Biotechnology) for overnight at 4 °C. After washing with TBST, the membranes were incubated with a 1 : 2000 dilution of secondary antibody (goat anti-mouse IgG, HRP conjugate, Millipore) for 1 h at room temperature. After washing with TBST, bands were visualized by treating the membranes with ImmunoStar LD (FUJIFILM) according to manufacturer's instructions and detecting the bioluminescence with LAS-4000 (GE).
6
Western Blot Analysis of Phospho-CREB
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Cytoplasmic extracts were prepared using Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) according to the manufacturer's instructions. Protein samples were electrophoresed in an Extra PAGE One Precast Gel (5% to 15%; Nacalai Tesque, Kyoto, Japan), transferred to a polyvinylidene difluoride membrane (Merck Millipore, Burlington, MA) at room temperature, and then incubated with Blocking One (Nacalai Tesque) for 1 hr. Antibodies against phospho‐CREB (Ser133), total CREB, and β‐actin were purchased from Cell Signaling Technology (Danvers, MA). After an overnight incubation, the blots were washed three times with a wash buffer (PBS with 0.2% Tween 20) for 15 min each time at room temperature and then incubated for 2 hr with a secondary horseradish peroxidase‐conjugated goat anti‐rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The blots were then washed three times as described above. Antigen detection was performed with an enhanced chemiluminescence system (GE Healthcare, Chicago, IL). Quantification of the Western blots was performed using FUSION SOLO S (Vilber‐Lourmat, Marne, France).
7
Calaxin Binding Assay with Axonemes
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Purified calaxin-/- sperm axonemes were incubated for 30 min on ice with or without 1 μM recombinant Calaxin protein in different salt concentration buffers, which were generated by adding 0, 50, 100, 150, 200, 250, or 300 mM of NaCl to HMDEKAc buffer containing 0.01% Nonidet P-40 and 5 μM BSA as a blocking agent. Axonemes were collected by centrifugation (10,000 g, 3 min) and dissolved in SDS sample buffer, followed by protein denaturation at 95 °C for 3 min. Proteins were separated by SDS-PAGE in 5–20% gradient gels (Extra PAGE One Precast Gel, Nacalai Tesque) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). After blocking with 5% skim milk (Nacalai Tesque) in TBST (Tris-buffered saline containing 0.1% Tween-20), membranes were incubated with primary antibodies of monoclonal anti-acetylated tubulin antibody (1:5000 dilution) and polyclonal antibodies (1:400 dilution), followed by several washes and incubation with peroxidase-conjugated secondary antibodies (1:5000 dilution). Blots were visualized by ECL Select Western Blotting Detection Reagent (GE Healthcare) and observed using a luminescent image analyzer (ImageQuant LAS4000mini, GE Healthcare). Blot signals were quantified by ImageJ/Fiji.
8
Annexin V Proteins Characterization
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The proteins were run on a 5–20% polyacrylamide gel (Extra PAGE one Precast gel, Nacalai Tesque) in Tris–glycine–SDS buffer, 200 V for 40 min and stained with Coomassie Brilliant Blue (CBB Stain One Super, Nacalai Tesque). The sizes of Annexin V, EGFP–Annexin V and mPlum–Annexin V calculated from the amino acid sequence were 39.8 kDa, 66.7 kDa and 65.5 kDa, respectively. Precision Plus Protein Standard (BIO-RAD) was used as a size marker.
9
RNA Isolation, RT-qPCR, and Western Blotting
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RNA was isolated from tissues and cells as previously described (60 (link)) and reverse transcribed using a High Capacity RNA-to-cDNA kit (Applied Biosystems). Real-time PCR was performed using the LightCycler system (Roche Diagnostics) with the primers described in SI Appendix, Table S2 . Regarding Western blotting, mouse tissues were suspended in radio-immunoprecipitation assay (RIPA) buffer [50 mM Hepes-NaOH (pH 7.6), 150 mM NaCl, 1 mM ethylene glycol tetraacetic acid (EGTA), 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), and 0.5% sodium deoxycholate] containing 1% protease inhibitor mixture (Nacalai Tesque), broken by sonication at 4 °C, and centrifuged at 20,000 × g for 30 min. The supernatants (1.0 to 8.5 μg of protein) were separated by 7.5% SDS-polyacrylamide gel electrophoresis (PAGE) (Extra PAGE One Precast Gel, Nacalai Tesque) and transferred to polyvinylidene difluoride (PVDF) membranes (Merck). After incubating at room temperature for 1 h in buffer A (PBS containing 0.05% Tween 20 and 1 to 5% skim milk), the membrane was incubated at 4 °C for 12 to 16 h with 0.2 μg/mL 4-C11 mAb in buffer A, followed by incubation for 1 h with 1 μg/mL HRP-goat anti-mouse IgG2a heavy-chain Ab. HRP activity was visualized using Western LightningPlus-ECL (PerkinElmer) and detected by LAS4000 (GE Healthcare).
10
Quantitative Immunoblotting of Neuronal Proteins
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Cultured neuronal cells at six to eight weeks were homogenized in RIPA buffer [50 mm Tris-Cl (pH 7.6), 150 mm NaCl, 0.5 mm EDTA, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, Complete protease inhibitor cocktail (Roche), 1 mm PMSF]. Equal amount (10–20 μg per lane) of proteins were separated in Extra PAGE One Precast Gel (Nacalai tesque Inc.) and transferred to PVDF membranes. The membranes were blocked in 5% nonfat milk/TBS for 1 h, and incubated with specific primary antibodies shown as below: rabbit anti-PS1 NTF (1:1000, G1Nr5; Sato et al., 2008 (link)), rabbit anti-PS1 CTF (1:1000, G1L3; Tomita et al., 1999 (link)), rabbit anti-PS2 CTF (1:5000, #ab51249, abcam), rabbit anti-APP (1:200, #18 961, IBL), mouse anti-N-cadherin (1:1000, #610920, BD Transduction), mouse anti-phospho tau (1:500, #MN1020, Millipore), rabbit anti-tau (1:1000, #A0024, DAKO), mouse anti-actin (1:10 000, #A1978, Sigma). The membrane was then incubated with IRDye 800CW or IRDye 680-labeled secondary antibodies (LI-COR Bioscience). Signals were developed and quantified with an Odyssey Infrared Imaging System (LI-COR Bioscience).
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