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8 protocols using np 40 lysis buffer

1

Kinase Assay for CK1γ1, CK1γ3, and RIPK3

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In vitro kinase assays were performed, as previously described34 , with some modifications. pCMV3-N-Flag-CK1γ1 and pCMV3-N-Flag-CK1γ3, or pcDNA3.1-hMLKL-Flag plasmids were transfected into HEK293T cells (per 15 cm dish: 20 µg plasmid + 55 µl PEI + 1 ml OptiMEM, incubate for 15-20 min at 25 °C). Cells were lyzed 48 h later in 0.75 ml of NP-40 lysis buffer (NLB) (25 mM HEPES (pH 7.5), 0.2% NP-40, 120 mM NaCl, 0.27 M sucrose, 2 mM EDTA, 2 mM EGTA, 50 mM NaF, 10 mM beta-glycerophosphate, 5 mM sodium pyrophosphate, 5 mM sodium orthovanadate (added fresh), 0.1% BME (added fresh), 1 mM PMSF (added fresh), 2X Complete protease inhibitor cocktail (Roche, added fresh). After centrifugation at 16,000x g, 15 min, 4 °C, lysates were incubated with anti-Flag-agarose beads for 4 h on a rotating wheel at 4 °C. The beads were washed twice with NLB containing phosphatase inhibitors (each wash for 5 min on a rotating wheel at 4 °C) and twice with a wash buffer containing 1% Triton X-100, 250 mM NaCl, 25 mM Hepes pH 7.4. Flag-tagged proteins were eluted with 0.2 mg/ml Flag peptide for 2 h at 4 °C, on a wheel. RIPK3 was purified from Rosetta™(DE3)pLysS (EMD Millipore) E. coli cells using the plasmid pGEX-4T-1-RIPK3 (http://www.addgene.org/78827/). GST-hRIPK3 was purified as above following E. coli lysis by sonication. Elution was made using 40 mM reduced glutathione in PBS.
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2

Protein Extraction and Immunoblotting

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Cells were lysed with NP-40 lysis buffer (Roche Diagnostics GmbH, Mannheim, Germany). Antibodies used were anti-GAPDH (ab8425), anti-RNF31 (ab46322) from Abcam, and anti-Myc (9E10), anti-ubiquitin (FL76), anti-HA antibody (F-7), anti-ERα (1D5 and HC-20) from Santa Cruz Biotechnology, Inc., Dallas, TX, USA.
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3

Western Blot Analysis of NRF2 Protein

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Cell were lysed using NP-40 lysis buffer (Roche, Basel, Switzerland) and the extracted proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride transfer membrane (Merck Millipore, Billerica, USA). The membrane was incubated with the appropriate anti-NRF2 and anti-GAPDH antibodies (Abcam, Cambridge, UK) overnight at 4 °C. The blots were developed with a peroxidase-conjugated secondary antibody and the proteins were visualized using by ECL Plus Detection Reagent (Merck Millipore, Billerica, USA). The gray-scale value was assessed by Gel-Pro analyzer.
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4

Western Blot Analysis of Signaling Proteins

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Antibodies were used to detect vimentin (Abcam, Cambridge, MA, and Cell Signaling, Danvers, MA), β-actin (Sigma, St. Louis City, MO), MMP2, MMP7, XIAP, cyclin D1, IL-1β, pSTAT3 (Y705), pSTAT3 (S727), STAT3, NF-κB (p65), p100/52, p105/50, pIkkα, Ikkα/β, pIkBα, IkBα (Santa Cruz Biotechnology), pJAK1, JAK1, pJAK2, JAK2 (Cell Signaling), GAPDH (Merck Millipore, Darmstadt, Germany), and IL-6 (Invitrogen, Carlsbad, CA). Antibodies, manufacturers, catalog numbers, and dilution for specific assays are summarized in Supplemental Table S1.
Cells were lysed with NP-40 lysis buffer supplemented with protease and phosphatase inhibitors (Roche, Manheim, Germany). Total proteins were separated by electrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane (GE Healthcare, Buckinghamshire, UK) as previously described [11 (link)]. The membrane was probed with each primary antibody at 4°C overnight and with HRP-conjugated secondary antibody (GE Healthcare) for 1 h at room temperature. The signals were detected using enhance chemiluminescence prime Western blotting detection kit (GE Healthcare). Image analysis was performed using Image Quant™ Imager and the band intensity was quantitated with ImageQuant TL software supplied by the manufacturer (GE Healthcare, Uppasala, Sweden). Three independent cultures were used for the analysis.
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5

Studying Wnt Signaling Protein Interactions

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Hek293t cells were co-transfected using linear PEI (1 mg/ml) with plasmids encoding HA-Gtpbp2, Myc, Myc-Gsk3b, and/or Myc-Axin. Cells were incubated overnight and lysed in NP40 lysis buffer (10 mM Tris-Cl pH 8.0, 137 mM NaCl, 10 % Glycerol, 1 % NP-40, + complete protease and phosphatase inhibitor tablets; Roche). Cell lysates were incubated with mouse anti-Myc (9E10) antibody overnight, purified using anti-IgG magnetic beads (NEB) according to the manufacturers instructions. Immunoprecipitations and whole cell lysates (1:10 of IP volumes) were blotted with mouse anti-myc (1:100) and rabbit anti-HA antibodies (1:500). For western blotting Xenopus embryos or explants were lysed in NP40 lysis buffer and extracted with an equal volume of 1,1,2-trichlorotrifluoro-ethane (Sigma), and then separated using SDS-page. Detection of western blots was conducted with IR secondary antibodies (Alexa 680 and IRdye800) scanned on a Licor Odyssey Classic Imager. Levels of ectopic myc-Axin were normalized to levels of co-injected HA-mCherry.
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6

Protein Extraction and Western Blot

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Cell pellets or fresh frozen tissue samples were lysed in 30 to 60 µl of NP-40 lysis buffer+protease inhibitors cocktail solution (Roche) and the protein phase was collected after centrifugation. Protein concentration was calculated using the Bradford Reagent Kit (BioRad). Absorbance readings were measured at 595 nm using a Beckman DU 530 Life Science UV/Visible spectrophotometer. After data collection, the concentration of the unknown samples was determined based on standard absorbance value. The protein samples were then exposed to SDS-polyacrilamide gel electrophoresis, transferred to a Hybond C super nitrocellulose membrane (GE Healthcare) and then blotted for the protein of interest. Membranes were washed and Enhanced Chemiluminescence (ECL) detection system (GE Healthcare) was used for visualization. The emitted fluorescence was detected using Hyperfilm ECL (GE Healthcare) on SRX-101A x-ray developer.
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7

Biotinylation and Internalization of VEGFR2

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HUVECs were grown to confluence and starved overnight in EBM2 with 0.5% FBS. Cells were rinsed, incubated with EZ-Link Sulfo-NHS-SS-Biotin (0.25 mg/ml, Thermo Scientific) at 4 °C for 1 h in PBS and rinsed with 50 mM glycine in PBS to stop the reaction. A portion of the cells were harvested and used to determine total biotinylated cell surface protein. The remaining cells were rinsed once with cold media +1% BSA, stimulated with EBM2 containing VEGF (25 ng/ml or 1.5 mM) or SLIT2 (1 μg/ml or 6 mM) at 37 °C for different times and then rinsed and incubated twice for 20 min each time on ice with the membrane-nonpermeable reducing agent GSH (45 mM, Sigma) in 75 mM NaCl, 75 mM NaOH, 1 mM EDTA, 1% BSA. GSH was quenched by incubating twice for 5 min each time with iodoacetamide (5 mg/ml) in PBS. Cell lysates were prepared using NP-40 lysis buffer (Roche). In total, 200 μg of protein from the cell lysate was immunoprecipitated with 50 μl of NeutrAvidin beads (Invitrogen) at 4 °C overnight, after which the beads were rinsed and resuspended in Laemmli SDS sample buffer. Samples were analyzed by SDS–PAGE followed by western blotting with anti-VEGFR2 antibody. The remaining cell lysates after bead incubation were used to blot VEGFR2, ROBO1, ENDOA2, and ACTIN loading controls.
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8

Quantifying HSV-1 Protein Expression

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Expression of the immediate-early protein (ICP0) and of a late protein (gD) of HSV-1 in U937 infected and mock-infected samples was evaluated, respectively, at 8 h and 16 h p.i. by western blotting. Cells were lysed in NP-40 lysis buffer containing protease inhibitors (Roche Applied Science, Penzerberg, Germany), and the lysates were electrophoresed through denaturing 10% SDS-polyacrylamide gel. Proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA), followed by block of non-specific binding with 5% non-fat milk in Tris-buffered saline (TBS) containing Tween 0.1%. Membrane was then incubated with primary antibodies, anti-gD, anti-ICP0 (Santa Cruz) and anti β-actin (Abcam), and then with HRP-conjugated secondary antibodies, followed by incubation with Pico-Sensitivity ECL (Thermo Scientific, Waltham, MA, USA). The ImageJ software was utilized to quantify densitometric values of blots [19 (link)].
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