Infected or transfected cells were lysed in NP-40 lysis buffer (50 mM HEPES pH 7.4, 150 nM NaCl, 1% (v/v) NP-40, supplemented with protease inhibitor cocktail [Sigma]), followed by centrifugation at 10,000 × g for 20 min to remove insoluble material. Cleared lysates were resolved on 7%, 10%, or 12% Bis-Tris SDS-PAGE gels (pH 6.4), and subsequently transferred to a Polyvinylidene difluoride (PVDF) membrane (Bio-Rad) using a Trans-Blot SD (Bio-Rad). Membranes were blocked with 5% (w/v) non-fat dry milk (NFDM) in PBS for 1 h, and then probed with primary antibody in antibody dilution buffer (PBS supplemented with 0.05% (v/v) Tween-20 and 5% (w/v) NFDM) at the indicated dilutions (as described above) for either 1 h at ~18–23 °C, or at 4 °C overnight. Membranes were probed with goat anti-mouse- or goat anti-rabbit-HRP antibodies (Cell Signaling cat #7076S and #7074S, respectively) in antibody dilution buffer for 1 h at ~18–23 °C. Proteins were visualized using an enhanced chemiluminescence reagent (Pierce) and detected by Fujifilm luminescent image analyzer LAS-4000 or Amersham Imager 600.
Las 4000
Manufactured by Cytiva
Sourced in United States
The LAS-4000 is a compact, high-performance imaging system designed for life science applications. It provides sensitive detection and quantification of proteins, nucleic acids, and other biological samples using chemiluminescence and fluorescence detection methods.
Automatically generated - may contain errors
Lab products found in correlation
Trans blot sd, by Bio-Rad (2 mentions)
Imager 600, by Cytiva (2 mentions)
Goat anti mouse or goat anti rabbit hrp antibodies, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Polyvinylidene difluoride pvdf membrane, by Bio-Rad (2 mentions)
Imager 680, by Cytiva (1 mentions)
Ecl prime kit, by Cytiva (1 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Ecl plus western blotting detection system, by Cytiva (1 mentions)
Ab211327, by Abcam (1 mentions)
Ab109224, by Abcam (1 mentions)
Phosstop, by Roche (1 mentions)
Immobilon, by Merck Group (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Protease inhibitor mixture, by Roche (1 mentions)
Bz x analyzer, by Keyence (1 mentions)
Bz x710, by Keyence (1 mentions)
Ab19016, by Merck Group (1 mentions)
Anti rabbit igg, by Cell Signaling Technology (1 mentions)
10 protocols using las 4000
1
Western Blot Protein Detection Protocol
2
Western Blot Protein Detection Protocol
3
Protein Expression Analysis After Drug Treatment
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500,000 cells were seeded in 6-well PO (1:500) and laminin (1:1000) coated plates 24h before commencing treatment. Cells were incubated in TC media supplemented with SAG (500nM), SANT-1 (500nM), JQ1 (500nM), INK128 (500nM), GDC-0084 (500nM) or AZD2014 (500nM) for 72h and lysed with 1x Cell Lysis Buffer supplemented with 1% SDS, 1:10 PhosSTOP and 1:100 Protease Inhibitor Cocktail. To analyze protein expression after lentiviral gene transfer, 500,000 cells were harvested and lysed as described above. A total 10-20μg of protein lysate was loaded onto 4%–12% Bis Tris gel, run for 55 min at 180V and 400mA in 1X MES running buffer and transferred to iBlot Gel Transfer Stacks. Membranes were blocked with 5% bovine serum albumin or 5% milk in 0.5% TBST (0.5% Tween 20 in Tris-Buffered Saline) and probed against beta-Actin (1:1000), Cleaved Caspase-3 (1:500), V5 tag (1:1000), AU1 tag (1:1000), p21 (1:1000), PARP (1:1000), Lamin B1 (1:1000), Oct4 (1:1000), 4E-BP1 (1:1000) and p-4E-BP1 (Thr37/46) (1:1000) overnight at 4 degrees centigrade. Signal was developed by probing the membranes with secondary HRP-conjugated anti-mouse or anti-rabbit antibodies diluted in blocking buffer (1:10,000) and incubating them with SuperSignal West Pico Chemiluminescent Substrate or SuperSignal West Femto Chemiluminescent Substrate. Signal was detected on ImageQuant LAS 4000 or an Amersham Imager 680.
4
Quantitative Western Blot Analysis
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Western blot analysis was performed as previously reported. A protein sample from a total cell extract (10 μg) was run on 10 % SDS-PAGE and transferred to a nitrocellulose membrane. The blot was then probed with the following primary antibodies: β-ACTIN (mouse IgG1, 1:5000, Sigma-Aldrich, USA), HB9 (mouse IgG1, 1:2000, DSHB, USA), ISL-1 (mouse IgG2a, 1:2000, DSHB, USA), and ChAT (goat IgG, 1:2000, Chemicon, USA). Signals were detected with HRP-conjugated secondary antibodies (Jackson ImmunoResearch laboratory Inc., USA) using an ECL Prime kit (Amersham Biosciences, USA). Quantitative analysis was performed using LAS4000 (Amersham biosciences, USA) and ImageJ software. The amount of protein loaded in each slot was normalized to β-ACTIN.
5
Protein Extraction and Immunoblot Analysis
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Proteins were extracted from cells using RIPA extraction and lysis buffer (Sigma) with the protease and phosphatase inhibitor cocktail (ThermoFisher Scientific). Protein concentrations were determined by BCA protein assay. Equal amounts of protein were loaded for immunoblot analysis. Signals were detected by the ECL plus Western blotting detection system (Amersham) and visualized by LAS-4000 lumino image analyzer.
6
Protein Extraction and Western Blot
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Cell lysates were prepared in radioimmunoprecipitation assay buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, and 1% NP-40) supplemented with Roche protease inhibitor cocktails (Cat# 4693132001, Sigma-Aldrich). SDS-PAGE separated equal amounts of total cell lysate. The amount of GAPDH (Cat# of anti-GAPDH is HRP-60004, Proteintech Group Inc.) served as a loading control. Primary antibodies against the following proteins were used in this manuscript: hMEX3B (Cat# WL3452579, Invitrogen), and HLA-A (Cat# DF6890, Affinity). Horseradish peroxidase-conjugated anti-rabbit or anti-mouse (Cat# STAR121 or AAC10P, Bio-Rad Laboratories Inc.) secondary antibodies were used to detect primary antibodies, and the signal was detected using LAS4000 (Cytiva Life Science).
7
Western Blot Analysis of Transcription Factors
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Cells were washed with phosphate‐buffed saline and then lysed in radioimmunoprecipitation assay buffer supplemented with protease inhibitor cocktail tablets (cOmplete; Roche Diagnostics) and phosphatase inhibitor tablets (PhosSTOP; Roche Diagnostics). Protein contents were quantified using XL‐Bradford (SDS‐PAGE Adapted) (Integrale). Lysates (10 μg) were resolved by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels and electrotransferred onto polyvinylidene difluoride membranes (Immobilon; Merck Millipore). After blocking membranes for 90 min at room temperature, they were incubated overnight at 4°C with one of the following primary antibodies (1:1000): rabbit monoclonal anti‐ASCL1 (ab211327; Abcam), rabbit monoclonal anti‐NEUROD1 (ab109224; Abcam), mouse monoclonal anti‐POU2F3 (sc‐293 402; Santa Cruz Biotechnology), and rabbit monoclonal anti‐YAP1 (14 074; Cell Signaling Technology). Hsp90 levels were used as a control for protein loading. After incubation with primary antibodies, membranes were incubated with a secondary antibody for 1 h at room temperature. Horseradish peroxidase‐linked anti‐rabbit (Cytiva) and anti‐mouse (Cytiva) antibodies were used as secondary antibodies. Proteins were detected by enhanced chemiluminescence (LAS‐4000; Cytiva).
8
Western Blot Analysis of c-Fos in NTS Cells
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The brain stem was dissected and the NTS cells isolated, to detect c-Fos expression using the western blot technique. Tissues were homogenized in a non-ionic detergent buffer (1% Nonidet-P40, 5% glycerol, 1 mM EDTA, 25 mM Tris-HCl, and 150 mM NaCl at pH 7.5) and supplemented with a protease inhibitor mixture (Roche Co., Basel, Switzerland) for 20 min at 4 °C. The lysates were centrifuged (21,130× g at 4 °C for 10 min) to obtain a protein supernatant. The protein concentration of each whole-cell extract was determined using the Bio-Rad Protein Assay Kit (Bio-Rad Laboratories Inc., Hercules, CA, USA). Equal amounts of protein from each protein extract (30 μg/lane) were separated by electrophoresis on an 8% sodium dodecyl sulfate-polyacrylamide gel and blotted onto PVDF transfer membranes. Epitope-specific primary antibodies including c-Fos and α-actin conjugated with secondary antibodies (Cell Signaling Technology Inc., Danvers, MA, USA) were used to label the target proteins. The bound antibodies were visualized with Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore Co., Billerica, MA, USA) and analyzed using a digital imaging system (LAS-4000; GE Healthcare Bio-Sciences Corp., Little Chalfont, UK).
9
Microscopic Analysis Using BZ-X710
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Microscopy images were taken by BZ-X710 and processed by BZ-X Analyzer (Keyence); pseudo-coloring was used as indicated in figure legends. For phase-contrast and fluorescence pictures, a representative section was chosen, cropped and magnified. Western blot data were recorded by LAS4000 (Cytiva). General image analysis was performed using Microsoft powerpoint as well as ImageJ v1.52.
10
Western Blot Analysis of Histones
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The samples were separated by 10% SDS-PAGE and transferred to PVDF membrane (Millipore). The membrane was incubated for 1 h with Blocking One (Nacalai Tesque) to block nonspecific binding. The membrane was then incubated overnight at 4 °C with antibodies against histone H2B (5HH2-2A8, Millipore), histone H1.2 (19649-1-AP, Proteintech), histone H2A (938CT5.1.1, Santa Cruz), histone H3 (1G1, Santa Cruz), histone H4 (L64C1, Cell signaling), RAGE (A-9, Santa Cruz), and MMP-9 (AB19016, Millipore) in 10% Blocking One-PBST, followed by the incubation for 1 h at room temperature with HRP-conjugated secondary antibodies (1:5,000 dilution, anti-mouse IgG, Cell signaling, #7076; 1:5,000 dilution, anti-rabbit IgG, Cell signaling, #7074). Detection was performed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and LAS4000 (Cytiva). The data were analyzed by ImageJ Fiji software version 1.52n. For the detection of total proteins, PVDF membrane was stained with 0.1% Ponceau S solution containing 5% acetate for 5 min. The stained membrane was scanned with LAS4000, destained with PBST, and then used for subsequent blocking and immunodetection for histone H2B.
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