For mouse muscles, ~10-20 mg of tissue was homogenized in NP40 buffer (Life Technologies) using zirconium beads in a NextAdvance bullet blender and the protein extracts were quantified. Protein samples were prepared by addition of SDS-Blue loading buffer and DTT (dithiothreitol; to a final concentration of 0.1 M) and heating at 95°C for 5 minutes. Samples were then run on 4%-20% gradient gels with a molecular weight ladder and transferred to PVDF membranes, which were blocked with either 5% milk powder or 5% BSA (according to manufacturer’s instructions) for 1 hour and then incubated with the primary antibodies listed in the Key Resources Table overnight at 4°C. After washing, the appropriate HRP-conjugated secondary antibodies were incubated at 4°C for 2 hours, washed again, and then probed with ECL reagents to detect the protein of interest. Ponceau S staining was also used to confirm even loading of protein samples.
Np 40 buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
NP-40 buffer is a non-ionic detergent used for cell lysis and protein extraction. It disrupts cell membranes while preserving protein structure and function.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (11 mentions)
Dynabead, by Thermo Fisher Scientific (7 mentions)
Nitrocellulose membrane, by Bio-Rad (6 mentions)
Bis tris gel, by Thermo Fisher Scientific (6 mentions)
Dynabeads, by Bio-Rad (6 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (6 mentions)
Laemmli sample buffer, by Bio-Rad (6 mentions)
Genemate blue ultra autoradiography film, by Avantor (5 mentions)
Complete protease inhibitor, by Roche (4 mentions)
Phosphatase inhibitor, by Roche (4 mentions)
Dc protein assay kit, by Bio-Rad (4 mentions)
Complete protease inhibitor cocktail, by Roche (3 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (3 mentions)
Protein g dynabead, by Thermo Fisher Scientific (3 mentions)
Ecl detection kit, by Thermo Fisher Scientific (3 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Horseradish peroxidase conjugated anti rat or anti mouse, by Agilent Technologies (2 mentions)
Enhanced chemiluminescence western blotting detection kit system, by Cytiva (2 mentions)
Pvdf membrane, by Cytiva (2 mentions)
51 protocols using np 40 buffer
1
Muscle Protein Extraction and Western Blot
2
Protein Expression Detection Protocol
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Animal tissues were shredded by TissueLyser LT (Qiagen, Hilden, Germany). The shredded tissues and cells were lysed in NP40 buffer (Life Technologies, Carlsbad, USA) containing protease inhibitor cocktail (Roche, Mannheim, Germany). Lysates were mixed with loading buffer and heated at 95 °C for 5 min. Protein samples were resolved on 4–12% SDS–PAGE gels (Life Technologies, Carlsbad, USA) and transferred onto nitrocellulose membranes (GE Healthcare, Freiburg, Germany). Non-specific antibody binding was blocked with 5% nonfat milk in TBST buffer (50 mM Tris/150 mM NaCl/0.1% Tween-20) for 1 h. The membranes were incubated with primary antibodies in incubation solution (2% milk in TBST) overnight. Myc-tag antibody (Cell Signaling #5605, Danvers, USA) was diluted 1:2500; Tubulin antibody (Sigma-Aldrich #T5168, Munich, Germany) was diluted 1:5000; RASAL antibody (Abcam #ab168610, Cambridge, UK; Biorbyt #orb101674, Cambridge, UK) was diluted 1:1000. The membranes were washed three times with 2% milk in TBST and incubated with HRP conjugated secondary antibody for 1 h. Membranes were visualized using LumiGLO chemiluminescence (Cell Signaling, Danvers, USA) and images were documented by a ChemiDoc MP System and processed using ImageLab software (Bio-Rad, Munich, Germany). All uncropped blots are included in Supplementary Fig. 13 .
3
Coimmunoprecipitation and GST Pull-down Assays
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Coimmunoprecipitations were performed as previously described (Kyei et al., 2009 (link)) with slight modification. In brief, cells were lysed with NP-40 buffer (Life Technologies) containing 1 mM PMSF and protease inhibitor cocktail (Roche) for 45 min followed by centrifugation. Supernatants were incubated for 2 h with antibodies at 4°C. The immune complexes were captured with Dynabeads (Life Technologies). Immunoprecipitates were washed three times with PBS, eluted with Laemmli SDS-PAGE sample buffer, and subjected to immunoblot analysis.
GST and GST-tagged proteins were expressed in Escherichia coli BL21 (DE3) or SoluBL21 (Amsbio). GST and GST fusion proteins were purified and immobilized on glutathione-coupled Sepharose beads (Glutathione-Sepharose 4 Fast Flow; GE Healthcare), and pull-down assays with in vitro translated [35S]-labeled proteins were performed as described previously (Pankiv et al., 2007 (link)). The [35S]-labeled proteins were produced using the TNT T7 Quick Coupled Transcription/Translation System (Promega) in the presence of [35S]l -methionine. The proteins were eluted from washed beads by boiling for 5 min in SDS-PAGE gel loading buffer, separated by SDS-PAGE, and radiolabeled proteins were detected in a bioimaging analyzer (BAS-5000; Fujifilm).
GST and GST-tagged proteins were expressed in Escherichia coli BL21 (DE3) or SoluBL21 (Amsbio). GST and GST fusion proteins were purified and immobilized on glutathione-coupled Sepharose beads (Glutathione-Sepharose 4 Fast Flow; GE Healthcare), and pull-down assays with in vitro translated [35S]-labeled proteins were performed as described previously (Pankiv et al., 2007 (link)). The [35S]-labeled proteins were produced using the TNT T7 Quick Coupled Transcription/Translation System (Promega) in the presence of [35S]
4
Glucose-Stimulated Insulin Secretion Assay
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Cells were seeded in a 24-well plate (0.3·106) 48 h prior to the experiment. Initially, the cells were pre-incubated at various concentrations (7–35 mM glucose in 40 mM phosphate buffer) for 2–8 h. The media were discarded, and the cells were incubated with 2 mM glucose in Krebs Ringer Hepes Buffer (KRHB) (NaCl (120 mM), KCl (5 mM), CaCl (2 mM), MgCl (1 mM), NaHCO3 (25 mM), HEPES (5.5 mM)) for 1 h. After this, GSIS was performed (1 h at 2 mM glucose, then 1 h 16.7 mM glucose in KRHB), all supernatants were collected for ELISA, and the cells were finally lysed with NP-40 buffer (Life Technologies) for insulin content measurements.
5
Muscle Protein Extraction and Western Blot
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For mouse muscles, ~10-20 mg of tissue was homogenized in NP40 buffer (Life Technologies) using zirconium beads in a NextAdvance bullet blender and the protein extracts were quantified. Protein samples were prepared by addition of SDS-Blue loading buffer and DTT (dithiothreitol; to a final concentration of 0.1 M) and heating at 95°C for 5 minutes. Samples were then run on 4%-20% gradient gels with a molecular weight ladder and transferred to PVDF membranes, which were blocked with either 5% milk powder or 5% BSA (according to manufacturer’s instructions) for 1 hour and then incubated with the primary antibodies listed in the Key Resources Table overnight at 4°C. After washing, the appropriate HRP-conjugated secondary antibodies were incubated at 4°C for 2 hours, washed again, and then probed with ECL reagents to detect the protein of interest. Ponceau S staining was also used to confirm even loading of protein samples.
6
Cloning and Transfection of LASP-1 in HA22T/VGH Cells
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The full lenght coding region (CDS) of LASP-1 was amplified from SKHep1C3 cDNA using the LASP-1 cl Forward: 5′-ggaaccatgaaccccaac-3′ and SH3 Reverse: 5′-cctccacgtagttggccg-3′ primers and then directly cloned in the pcDNA3.1-CT-GFP-TOPO vector (Life Technologies) upstream the GFP gene following manufacturer’s instructions. The correct sequence and the orientation of the insert were ascertained by direct automatic sequencing of the plasmid. The vector, named pGFP-LASP1, was transiently transfected in HA22T/VGH cells. Briefly, 2,900,000 HA22T/VGH cells were seeded in 10-cm diameter Petri dishes and 24 h later, when the cells reached 80% confluency, they were transfected with 24 μg/dish of pGFP-LASP1 plasmid using 60 μl/dish of Lipofectamine (Life Technologies) following the manufacturer’s instructions. After 72 h of transfection, cell lysates were collected for immunoprecipitation analysis in NP-40 buffer (Life Technologies) containing phosphatase (Sigma) and protease (Roche) inhibitor cocktails or in 0.05% SDS for routine analysis by western blotting (WB).
7
Preparation of Cell Media and Lysates
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Both HEK Flp-In and HEK293T cells were grown to confluence in a 100 mm dish format. After washing with Dulbecco’s Phosphate-buffered saline (PBS), cell medium was replaced by serum-free OPTIMEM I medium (Life Technologies). Cell media and cell lysates were harvested 24 h later. One ml of cell medium, supplemented with the protease inhibitor AEBSF (1 mM final, Calbiochem), was centrifugated (15.000 g for 5 min, 4 °C). Supernatants were aliquoted and immediately stored at −80 °C. After an initial wash with PBS, cell lysates were prepared by collecting the cells with a cell scraper followed by lysis with 2 ml NP40 buffer (Life Technologies) supplemented with the protease inhibitor AEBSF. The cell lysates were incubated on ice for 10 min, centrifugated (15.000 g for 5 min, 4 °C) and immediately stored at −80 °C.
8
TLR4 Signaling Complex Characterization
9
Western Blot Analysis of Protein Expression
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Cells were lysed in NP40 buffer (Biosource, Invitrogen, Paisley, UK). Samples containing equal amounts of protein were electrophoresed under reducing conditions in 8–10% SDS-PAGE gels. Protein was electroblotted to polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences, Buckinghamshire, UK). Blots were probed against primary antibodies of interest and horseradish peroxidase-conjugated anti-rat or anti-mouse (Dako) were used as secondary antibodies. Bound antibodies were detected with the enhanced chemiluminescence Western blotting detection kit system (Amersham). Blots were probed for HSC70 (Santa Cruz Biotechnology) as a loading control.
10
Western Blot Analysis of MCT1 Protein
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Cells were lysed in NP40 buffer (Biosource, Invitrogen, Paisley, UK). Samples containing equal amounts of protein were electrophoresed under reducing conditions in 8–10% SDS-PAGE gels. Protein was electroblotted to PVDF membranes (Amersham Biosciences, Buckinghamshire, UK). Blots were probed with antibodies against MCT1 (Santa Cruz Biotechnology). Horseradish peroxidase-conjugated anti-rat or anti-mouse (Dako) was used as secondary antibodies. Bound antibodies were detected with the enhanced chemiluminescence western blotting detection kit system (Amersham). Blots were probed for Total FAK (Millipore) as a loading control.
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