The protocol was adapted from Bjedov et al. (2010) (link). Briefly 6 pairs of ovaries were freshly dissected in cell insect media and homogenized in 30 μl of 4x Laemmli loading sample buffer (Invitrogen, #NP0008) containing 10x sample reducing agent (Invitrogen, #NP009). Extracts were cleared by centrifugation and boiled for 10 min at 90°C. 10 μl of protein extract was loaded per lane on polyacrylamide gel (Invitrogen, #NP0335). Proteins were separated and transferred to nitrocellulose membrane. Primary antibodies used were as follows: guinea pig anti-dS6K (gift of Aurelius Teleman,1:5,000,) (Hahn et al., 2010 (link)) and rabbit anti-phospho-Thr398-S6K (Cell Signaling Technologies #9209, 1:1,000). HRP- conjugated secondary antibodies (Jackson Immunoresearch, AffiniPure anti-rabbit #111-005-144 and anti-guinea pig #106-005-003) were used. Blots were developed using the ECL detection system (PerkinElmer, #NEL105001EA). Western blots were analyzed using ImageJ program (US National Institutes of Health).
Nel105001ea
Manufactured by PerkinElmer
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The NEL105001EA is a laboratory instrument designed for the detection and quantification of various analytes. It utilizes advanced spectroscopic techniques to perform sensitive and accurate measurements. The core function of this product is to provide researchers and scientists with a reliable tool for analytical applications.
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Lab products found in correlation
Ipvh00010, by Merck Group (5 mentions)
Dc protein assay kit, by Bio-Rad (3 mentions)
Quantity one software, by Bio-Rad (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
A1978, by Merck Group (3 mentions)
Chemidoc xrs system, by Bio-Rad (3 mentions)
G9917809, by Cytiva (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
A2856, by Merck Group (2 mentions)
Sc 374353, by Santa Cruz Biotechnology (2 mentions)
Sc 515791, by Santa Cruz Biotechnology (2 mentions)
Sc 22597, by Santa Cruz Biotechnology (2 mentions)
Ab108327, by Abcam (2 mentions)
Ab170952, by Abcam (2 mentions)
Sc 271430, by Santa Cruz Biotechnology (2 mentions)
Protein a agarose beads, by Cell Signaling Technology (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Gel pro analyzer 4, by Media Cybernetics (1 mentions)
Lysis buffer, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
24 protocols using nel105001ea
1
Ovary Protein Extraction and Western Blot
2
Immunoblotting and Immunoprecipitation Protocols
3
Protein Extraction and Western Blot Analysis
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The cells and tissue samples were homogenized in lysis buffer (50 mM pH 7.5 HCl-Tris, 300 mM NaCl, 0.5% SDS, and 1% Triton X-100) and incubated 15 min at 95°C. Protein concentration of the extracts was measured using the DC protein assay kit (500–0111, Bio-Rad). Equal amounts of total protein extract from healthy and SAD cells were resolved by SDS-PAGE and then transferred to nitrocellulose (G9917809, Amersham, Germany) or PVDF (IPVH00010, Merk Millipore, Cork, Ireland) membranes. Western blot and immunoreactive proteins were developed using an enhanced chemiluminescence detection kit (NEL105001EA, Perkin Elmer) following instructions of the supplier. Quantification was performed by densitometry of the obtained bands in each lane with respect to the correspondent housekeeping protein such as GAPDH or β-tubulin band in each experiment (Quantity One software, Bio-Rad).
4
Western Blot Analysis of Protein Extracts
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The harvested cells were lysed in ice for 30 min with lysis buffer (Cell Signaling Technology, 9803). Lysates were cleared by centrifugation at 14,000 rpm for 10 min at 4 °C. Protein concentrations were measured by the Bradford assay (Bio-Rad Laboratories, 500–0006). For Western blot analysis, cell lysates were boiled for 5 min with sample buffer before being resolved in SDS–polyacrylamide gels. The proteins were transferred to PVDF membrane (Millipore, IPVH00010). The membrane was blocked with 5 % skim milk in TBST buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1 % Tween 20) for one hour and then stained with primary antibodies at 4 °C overnight followed by incubation with secondary antibodies (antibodies are listed in Additional file 7 : Table S1). The binding of each antibody was detected using an enhanced chemi-luminescence kit (PerkinElmer, NEL105001EA). The signals were detected by X-ray films (Fuji, 47410 08399) or a UV transilluminator (UVP Ltd., BioSpectrum™ 500 Imaging System) and analyzed by the Gel-Pro Analyzer 4.0 software (Media Cybernetics).
5
Protein Expression Analysis in Cells
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The cells or tissues were lysed using radioimmunoprecipitation assay buffer (Solarbio, Beijing, China). The lysis solution was collected after centrifugation at 12 000 × g for 10 minutes at 4°C. The protein concentration was determined using bicinchoninic acid assay. Total protein (20 μg) was subjected to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% skimmed milk and then incubated with a primary antibody overnight at 4°C. After washing, the membranes were incubated with HRP‐conjugated secondary antibody according to the manufacturer's protocol. The antibodies used in this study were as follows: rabbit anti‐apelin (1:2000, Cat. No. ab59469), rabbit anti‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH, 1:3000, Cat. No. ab9485), and goat anti‐rabbit immunoglobulin G (HRP, 1:5000, Cat. No. ab6721; Abcam, Cambridge, UK). An ECL detection system (NEL105001EA, PerkinElmer, Waltham, MA, USA) was used to visualize the specific bands.
6
Protein Extraction and Immunoblotting Protocol
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Cells were lysed in 100 μl lysis buffer (20 mM Tris-HCl, pH 7.5, 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, 100 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 10 mM sodium orthovanadate, and 2 mM PMSF) with protease inhibitor cocktail (MDBio, 2043). Sodium pyrophosphate and β-glycerophosphate were used to be the false substrates for phosphatase in general. For immunoblotting, the total protein content was determined using the Bradford assay. Total cell lysates were mixed with 1/4 volume of sample buffer (250 mM Tris-HCl, 500 mM DTT, 10% SDS, 0.1% bromophenol blue, and 50% glycerol) for 5 min at 95 °C and separated by SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes (Millipore, IPVH00010). After blocking with 5% nonfat milk in PBS-T (0.05% Tween-20), blots were washed with PBS-T for three times, then probed with primary antibodies at 4 °C overnight. After PBS-T washing, blots were stained with HRP-conjugated secondary antibodies in 1/5000 dilution at room temperature for 1 h. Blots were developed using Western lightning chemiluminescence reagent (Millipore, WBKLS0500; PerkinElmer, NEL105001EA).
7
Cell Lysis and Protein Quantification
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Cells were seeded in Petri dishes at the density of 35,000–40,000 cells/cm2 and treated as indicated when confluence reached approximately 80%. Cell homogenates were prepared by freeze-thawing and ultrasonication in RIPA lysis buffer containing protease inhibitors as described in Ref. 21 (link). The blots were detected using enhanced chemiluminescence reagents (ECL, cod. NEL105001EA; PerkinElmer, Waltham, MA) and developed with the ChemiDoc XRS instrument (BioRad, Hercules, CA, USA). Intensity of the bands was estimated by densitometry using Image Lab 6.0 (BioRad, Hercules, CA, USA).
8
Western Blot Analysis of Muscle Protein Signaling
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Western blot assays were carried out with gastrocnemius muscles or C2C12 cells homogenized in RIPA buffer [50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1% NP‐40, 0.5% sodium deoxycholate, and 0.1% SDS]. Proteins were separated on a 4–12% Bis‐Tris gel (Life Technologies) and transferred onto PVDF membranes. The membranes were incubated with the following primary antibodies at 4°C overnight: anti‐IGF1 receptor (IGF1R) (1:1000; sc713, Santa Cruz), anti‐eIF4E (1:2000; sc9976, Santa Cruz), anti‐phospho‐eIF4E(Ser209) (1:2000; 9741, Cell Signaling), anti‐4EBP1 (1:2000; 9452, Cell Signaling), anti‐phospho‐4EBP1(Ser65) (1:1000; 9451, Cell Signaling), anti‐AKT (1:2000; 9272, Cell Signaling), anti‐phospho‐AKT (Ser473) (1:2000; 4060, Cell Signaling), anti‐puromycin (1:25000; MABE343, Sigma), anti‐eIF3M (1:1000; NBP1‐31045, Novus Biologicals), anti‐eIF4G (1:2000; 2498, Cell Signaling), anti‐eIF4B (1:1000; 3592, Cell Signaling), anti‐eIF2B5 (1:1000; 3595, Cell Signaling), and anti‐phospho‐eIF2α (Ser51) (1:1000; 9721, Cell Signaling). The membranes were next incubated with horseradish peroxidase‐conjugated secondary antibodies to the appropriate IgG (1:2000; Life Technologies) for 90 min. Bound antibodies were visualized with enhanced chemiluminescence reagents (NEL105001EA, PerkinElmer).
9
Western Blot Protein Analysis Protocol
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Proteins were extracted using RIPA Lysis Buffer (P0013, Beyotime, China) and quantified using a BCA kit (P0009, Beyotime, China). Fifty micrograms of each protein sample was separated by 8, 10, or 15% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 5% BSA and incubated with primary antibodies for 10 h at 4 °C. The membranes were rinsed five times with PBS containing 0.1% Tween 20 and incubated for 1 h with the appropriate horseradish peroxidase-conjugated secondary antibody at 37 °C. Membranes were extensively washed with PBS containing 0.1% Tween 20 three times. The signals were stimulated with enhanced chemiluminescence substrate (NEL105001 EA, PerkinElmer) for 1 min and detected with a Bio-Rad ChemiDoc MP System (170–8280). The primary images (Supplementary Figs. 9 -11 ) were cropped for presentation.
10
Western Blot Analysis of Urea Cycle Enzymes
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Tissues and tumor samples were grounded in liquid nitrogen, lysed in Tris lysis buffer (50 mM Tris HCl, 150 mM NaCl, 1 mM EDTA, 0.1% NP40, 5 mM MgCl2, 10% glycerol), separated on 12.5% SDS-PAGE gel and then transferred on PVDF membrane (Millipore). Membranes were blocked with 5% non-fat milk for 1 hour and probed overnight at 4˚C with antibodies against ASS1 (1:1,000, Ab170952, Abcam), ASL (1:500, sc-374353, Santa Cruz), OTC (1:500, sc-515791, Santa Cruz), ARG1 (1:500, sc-271430, Santa Cruz), ATG7 (1:2,000, A2856, Sigma), transferrin (1:1,000, sc-22597, Santa Cruz), ATG5 (1:1,500, Ab108327, Abcam) and β-actin (1:5,000, A1978, Sigma). Immunoreactive bands were detected using peroxidase-conjugated antibody (GE Healthcare) and enhanced chemiluminescence detection reagents (NEL105001EA, Perkin Elmer) and were analyzed using the ChemiDoc XRS+ system (Biorad). Protein levels were quantified using the Image Lab v6.0.1 software. Antibodies were validated with the use of positive and negative control following manufacturer’s protocol.
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