Cells were lysed in standard lysis buffer, sonicated and cleared by centrifugation. Protein separation was performed on 4–12% BisTris NuPAGE pre-cast gels (ThermoFisher Scientific AG) and transferred to nitrocellulose membranes (GE Healthcare, Glattbrugg, Switzerland). After 1 h of blocking in 5% milk in 0.2% PBS-Tween, membranes were incubated with primary antibodies over night at 4 °C and for 2 h with HRP-linked secondary antibody at room temperature. Protein detection was carried out by chemiluminescence using ECL detection reagent (GE Healthcare) or SuperSignalTM Western blotting reagent (ThermoFisher Scientific AG). Quantification of blots was performed using ImageJ (version 1.46r), Student t test *p < 0.05 if indicated, analyzed by Prism GraphPad. Full-length blots of figure blots are presented in Supplementary Fig. S6 .
Bistris nupage pre cast gels
Manufactured by Thermo Fisher Scientific
Sourced in United States
BisTris NuPAGE pre-cast gels are laboratory equipment used for protein electrophoresis. They are pre-cast polyacrylamide gels designed for the separation and analysis of proteins.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (3 mentions)
Ecl detection reagent, by GE Healthcare (2 mentions)
Bradford protein assay, by Bio-Rad (2 mentions)
Mini trans blot electrophoretic transfer cell, by Bio-Rad (2 mentions)
Odyssey image studio software, by LI COR (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Supersignal west dura ecl substrate, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Chemidoc imager, by Bio-Rad (1 mentions)
Protein a bead, by Thermo Fisher Scientific (1 mentions)
Image lab version 6, by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Complete mini protease inhibitor cocktail, by Merck Group (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Pvdf fl membrane, by Merck Group (1 mentions)
X tremegene 9 dna transfection reagent, by Roche (1 mentions)
9 protocols using bistris nupage pre cast gels
1
Western Blot Protein Detection Protocol
2
Protein Extraction and Western Blot
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Cells were trypsinized and harvested in the appropriate growth medium and lysed on ice for 10 min in protein lysis buffer (PLB) (10 mM HEPES pH 7.0, 0.1 M KCl, 5 mM MgCl2, 25 mM EDTA pH 8.0, 0.5% (v/v) NP-40, Proteinase Inhibitor, 20 µM DTT), followed by centrifugation at 16,000 x g for 15 min. The concentrations of the lysates were measured using Bradford Protein Assay (Bio-Rad). 8–12 µg of protein lysates was separated on 4–12% Bis-Tris NuPAGE® Precast gels (ThermoFisher Scientific) and transferred to PVDF membranes using the Mini Trans-Blot® Electrophoretic Transfer Cell (BioRad) in transfer buffer (2.5 mM Tris, 19.2 mM glycine and 10% (v/v) methanol). The membranes were probed with specific primary antibodies followed by the corresponding secondary antibodies.
3
Protein Expression Analysis by Western Blot
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Cells and tissues were lysed in RIPA buffer (ThermoFisher Scientific) containing protease inhibitor cocktail (ThermoFisher Scientific). Samples were centrifuged and supernatant collected for protein analysis. After protein quantification with the BCA method, samples were separated using Bis-Tris NuPage precast gels (ThermoFisher Scientific) and transferred to PVDF membranes. After blocking with 5% nonfat milk for 1 hour, primary antibodies were incubated overnight at 4°C. Membranes were washed in TBS-tween buffer and incubated in HRP conjugated secondary antibodies for 1h at room temperature. Blots were developed using SuperSignal West Dura ECL substrate (ThermoFisher Scientific) and imaged using FluorChem M ProteinSimple imager.
4
Protein Extraction and Western Blotting
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Whole-cell lysates were sonicated in ice-cold radioimmunoprecipitation assay buffer freshly supplemented with protease inhibitors (0.05 M Hepes [pH 7.4], 1% Triton X-100, 0.1% SDS, 50 mM sodium fluoride, 0.05 M EDTA, 5% sodium deoxycholate, 1 mM sodium orthovanadate, and Protease Inhibitor Cocktail [Sigma]). Total protein was quantified by bicinchoninic acid assay, as per the manufacturer’s instructions (Thermo Scientific).
Lysates were resolved in reducing conditions on 4 to 12% gradient Bis–Tris NuPAGE precast gels (Thermo Scientific) before wet transfer to nitrocellulose. Blots were probed with primary antibodies MTOR (4517S), pMTOR S2448 (5536S), Bruton’s tyrosine kinase (BTK) (8547S), pBTK Y223 (5082S), ERK1/2 (9107S), or pERK1/2 T202/Y204 (mouse T203/Y205, 9101S) (Cell Signaling Technology) followed by horseradish peroxidase–conjugated mouse or rabbit secondary antibody. Blots were developed using Immobilon Classico ECL substrate (Millipore) before visualization on a Chemidoc imager (Bio-Rad). Densitometry was performed using Image Lab (Bio-Rad).
Lysates were resolved in reducing conditions on 4 to 12% gradient Bis–Tris NuPAGE precast gels (Thermo Scientific) before wet transfer to nitrocellulose. Blots were probed with primary antibodies MTOR (4517S), pMTOR S2448 (5536S), Bruton’s tyrosine kinase (BTK) (8547S), pBTK Y223 (5082S), ERK1/2 (9107S), or pERK1/2 T202/Y204 (mouse T203/Y205, 9101S) (Cell Signaling Technology) followed by horseradish peroxidase–conjugated mouse or rabbit secondary antibody. Blots were developed using Immobilon Classico ECL substrate (Millipore) before visualization on a Chemidoc imager (Bio-Rad). Densitometry was performed using Image Lab (Bio-Rad).
5
Protein Extraction and Western Blot
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Cells were trypsinized and harvested in the appropriate growth medium. Cells were lysed on ice for 10 minutes in protein lysis buffer (10 mmol/L HEPES pH 7.0, 0.1 mol/L KCl, 5 mmol/L MgCl 2 , 25 mmol/L EDTA pH 8.0, 0.005% (v/v) NP-40, 2 mmol/L DTT, Proteinase Inhibitor) and the lysates were centrifuged at 16,000 Â g for 15 minutes. The supernatants containing proteins were collected and the concentrations were measured using Bradford Protein Assay (Bio-Rad). A total of 8-15 mg of protein lysates was separated on 4%-12% Bis-Tris NuPAGE Precast gels (Thermo Fisher Scientific) and transferred to polyvinylidene difluoride membranes using the Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) in transfer buffer [25 mmol/L Tris, 192 mmol/L glycine, and 20% (v/v) methanol]. The membranes were probed with specific primary antibodies followed by the appropriate secondary antibodies.
6
Immunoprecipitation of Oligomeric Aβ
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Two micrograms of oAβ1–42 were added into 400 μL of 1% solution of Tween-20 in TBS (1% TBST) with protease inhibitor cocktail. Two micrograms of sTREM2-Fc, sTREM1-Fc or Fc protein was pre-bound to 25 μL of protein A beads (Thermo Fisher Scientific, 20,334). The beads were then incubated with oAβ1–42 in 1% TBST at 4 °C overnight. Beads were washed 5 times with 1% TBST for 3 min, resuspended in 20 μL 2 × SDS loading buffer and subjected to electrophoresis on 4–12% Bis-Tris NuPAGE precast gels (Thermo Fisher Scientific, NP0322PK2).
7
Immunoblotting Protocol with Optimized Lysis and Detection
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Cells were lysed in a modified RIPA buffer (50 mM Tris–Cl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 1 mM EGTA, 0.1% SDS, 50 mM NaF, 10 mM sodium β-glycerolphosphate, 5 mM sodium pyrophosphate, and 1 mM sodium orthovanadate and supplemented with Complete Mini Protease Inhibitor cocktail (Merck)), flash-frozen in liquid nitrogen and cleared via centrifugation. The samples were run for separation on 4–12% BisTris NuPAGE pre-cast gels (ThermoFisher Scientific) and transferred to nitrocellulose membranes by Trans-Blot Turbo Transfer System (BioRad). The membranes were then blocked for 1 h in 5% milk or BSA in TBS-T (100 mM NaCl, 10 mM Tris, pH 7.6, 0.1% Tween 20) and cut prior to incubation with primary antibodies at appropriate dilution overnight at 4 °C. After washing with TBS-T the membranes were incubated in corresponding horseradish peroxidase (HRP)-linked secondary antibody at room temperature for 1 h. Proteins on the membranes were detected via chemoluminescence induced by ECL detection reagent (GE Healthcare) or SuperSignal Western blotting reagent (ThermoFisher Scientific) using the ChemiDoc Imaging system (Biorad). The blots were quantified using Image Lab version 6.1 software (BioRad) and the data were analyzed using Prism GraphPad version 8. Full-length blots of blots used in figures are presented in Supplementary Fig. S7 .
8
Western Blot Analysis of GABAA Receptors
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The HEK293T cells were collected in modified radioimmunoprecipitation assay (RIPA) buffer (50 mM of Tris (pH = 7.4), 150 mM of NaCl, 1% NP-40, 0.2% sodium deoxycholate, 1 mM of EDTA) and 1% protease inhibitor cocktail (Sigma-Aldrich, Saint Louis, MO, USA). The collected samples were subjected to gel electrophoresis using 4–12% BisTris NuPAGE precast gels (Invitrogen) and transferred to polyvinylidene difluoride fluorescence-based (PVDF-FL) membranes (Millipore, Burlington, MA, USA). The primary antibodies used to detect GABAA receptors were as follows: Mouse α1 subunit antibody (1:500; NeuroMab, 75–136), rabbit β3 subunit antibody (1:500; Novus, NB300-199), and rabbit γ2 subunit antibody (1:500; Millipore, AB5559). The Mouse anti-Na+/K+ ATPase antibody (1:000; DSHB, a6F) was used to determine the density of the loading control. IRDye® (LI-COR Biosciences, Lincoln, NE, USA) conjugated secondary antibody was used at a 1:10 000 dilution in all cases. The blotted membranes were scanned using the Odyssey Infrared Imaging System (LI-COR Biosciences). The band-of-interest integrated intensity values were determined using the Odyssey Image Studio software (LI-COR Biosciences). Biotinylation protocols were described previously [23 (link)].
9
Kv3.2 Protein Expression and Analysis
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To detect the Kv3.2 protein expression and to perform protein functional analysis, the WT and two mutated cDNA plasmids were transfected to CHO stable cells (ATCC, USA) by X-tremeGENE 9 DNA Transfection Reagent (Roche). The transfected cells were collected and lysed in modified radio-immunoprecipitation assay lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1% NP-40, 0.2% sodium deoxycholate, 1 mM EDTA), and 1% protease inhibitor cocktail (Sigma-Aldrich, USA). Collected protein samples were subjected to gel electrophoresis using 4%–12% BisTris NuPAGE precast gels (Invitrogen Life Technologies, USA) and transferred to PVDF-FL membranes (MilliporeSigma, USA). Primary antibody against FLAG epitope tag located on FLAG fusion proteins (Sigma-Aldrich, polyclonal ANTI-FLAG, rabbit host. F7425) was used to detect the Kv3.2 protein by indirect immunofluorescent staining at a 1:500 dilution. Anti-Na+/K+ ATPase antibody (Developmental Studies Hybridoma Bank, antibodies at the University of Iowa for use in research, USA) at a 1:1,000 dilution was used as an internal quality control. IRDye conjugated secondary anti-rabbit antibody (LI-COR Biosciences, USA) was used at a 1:10,000 dilution. The membranes were scanned using the Odyssey Infrared Imaging System, and the integrated density value of bands was determined using the Odyssey Image Studio software (LI-COR Biosciences, USA).
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