The total protein concentrations in the cell lysates were quantified and equivalent amounts of cell proteins from two independent biological replicates were diluted in SDS-PAGE loading buffer containing 5% β-mercaptoethanol prior to boiling, separated using one-dimensional SDS-PAGE (4% to 12% Novex Bis-Tris mini gel, Invitrogen), and then visualized by Coomassie blue stain (Pierce). The entire protein gel lane was excised in parallel and cut into 48 gel slices each. The gel slices were subsequently subjected to in-gel digestion with trypsin (20 ng/μL) at 37°C overnight, as described previously [13 (link), 14 (link)]. The resulting tryptic peptides were extracted by 2.5% formic acid and 90% acetonitrile, lyophilized in a SpeedVac (Helena Biosciences), and resuspended in 0.1% formic acid and 2% acetonitrile before LC–MS/MS analysis.
Bis tris novex mini gel
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The Bis-Tris Novex mini-gel is a polyacrylamide gel used for protein electrophoresis. It is designed to provide high-resolution separation of proteins in a neutral pH environment.
Automatically generated - may contain errors
Lab products found in correlation
Simplyblue safestain, by Thermo Fisher Scientific (4 mentions)
Trypsin, by Promega (3 mentions)
Coomassie blue, by Bio-Rad (2 mentions)
Xcell surelock mini cell system, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap xl, by Thermo Fisher Scientific (1 mentions)
Coomassie staining, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap velos, by Thermo Fisher Scientific (1 mentions)
Qubit fluorometry, by Thermo Fisher Scientific (1 mentions)
Instantblue, by Abcam (1 mentions)
20 protocols using bis tris novex mini gel
1
Gel-based Proteome Fractionation and Tryptic Digestion
2
SDS-PAGE Analysis of Protein Samples
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We ran SDS-PAGE gels of triplicate samples (50 µg each technical replicate) on a 1 mm 10% NuPAGE® Novex® Bis-Tris Mini Gel using the XCell SureLock Mini-Cell system (Invitrogen) as per manufacturer's instructions for reduced samples. The gel was then stained using SimplyBlue™ SafeStain (Invitrogen), and destained following the manufacturer's instructions. The three lanes were then separated from the gel and cut into 16 sections of equal size. Next, half of each section was transferred to a 96-well plate, resulting in a total of 48 gel sections and stored at -80 °C until later analysis.
3
HeLa Cells Luciferase Fusion Assay
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HeLa cells transfected with the G.luciferase-MrgprC11 fusion construct were treated with cat S (2 µM) or papain (0.02 µM) for 10 minutes. Cells were pelleted and the supernatant was added directly to loading buffer to quench enzymatic activity, run on NuPAGE Novex Bis-Tris mini gels and transferred to nitrocellulose membranes. Membranes were probed with a primary rabbit anti-G. luciferase antibody followed by HRP-labeled donkey anti-rabbit antibody for reaction with the ECL-substrate to identify protein bands. The results presented are representative of one of four experiments.
4
HeLa Cells Luciferase Fusion Assay
5
Gel-Based Protein Separation and Mass Spectrometry
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Each sample was separated on a 10% Bis‐Tris Novex® mini‐gel (Life Technologies) using the 2‐(N‐morpholino)ethanesulfonic acid buffer system. The gel was stained with Coomassie and each lane excised into ten equally sized segments. Gel pieces were robotically processed (ProGest, DigiLab, Hopkinton, MA, USA) with the following protocol: (i) washed with 25 mM ammonium bicarbonate followed by acetonitrile, (ii) reduced with 10 mM dithiothreitol at 60°C followed by alkylation with 50 mM iodoacetamide at 20℃, (ii) digested with trypsin (Promega) at 37°C for 4 h and quenched with formic acid. The supernatant was analyzed directly without further processing.
6
Protein Reduction, Alkylation, and Tryptic Digestion
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Proteins were reduced in 10 mM DTT, alkylated in 50 mM iodoacetamide and incubated at 95 °C for 5 min in 1X Laemmli buffer. They were then separated by one-dimensional SDS-PAGE (4–12% Bis-Tris Novex mini-gel, Life Technologies) and visualized by Coomassie staining (Simply Blue Safe Stain, Life Technologies). Following extensive washes in water, the gel was cut into slices and subjected to in-gel digestion with 12.5 ng/ml trypsin (Promega) in 20 mM NH4HCO3. Tryptic peptides were extracted by 1% formic acid, then 100% acetonitrile. Solvent was removed by lyophilization in a speed vacuum centrifuge, and the tryptic peptides were resuspended in 1% formic acid.
7
Protein Separation and Tryptic Digestion
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For each time point, proteins were reduced in 10 mm DTT and alkylated in 50 mm iodoacetamide prior to boiling in loading buffer, and then separated by one-dimensional SDS-PAGE (4–12% Bis-Tris Novex mini-gel, Life Technologies) and visualized by Coomassie staining (Simply Blue Safe Stain, Life Technologies). The entire protein gel lanes were excised and cut into 8 slices each. Every gel slice was subjected to in-gel digestion with trypsin (23 (link)). The resulting tryptic peptides were extracted by 1% formic acid, then 100% acetonitrile, lyophilized in a speedvac, and resuspended in 1% formic acid.
8
Protein Identification by Mass Spectrometry
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Protein samples generated by the pulldowns were separated by one-dimensional SDS–PAGE (4–12% bis-Tris Novex mini-gel, Invitrogen). The resulting separated proteins were cut from the gel in six slices and subjected to in-gel digestion with trypsin. Trypsin digested peptides were separated using an Ultimate U3000 nanoflow LC-system (Dionex Corporation) consisting of a solvent degasser, micro and nanoflow pumps, flow control module, UV detector and a thermostated autosampler. The HPLC system was coupled to a LTQ Orbitrap XL (Thermo Fisher Scientific Inc.) via a nano ES ion source (Proxeon Biosystems). Quantification was performed with MaxQuant version 1.0.7.4 (Cox and Mann, 2008 (link)).
9
Protein Fractionation and Digestion
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Equal amounts of protein from labelled samples (1:1) were combined prior to protein digestion. Briefly, samples were reduced in 10 mM DTT and alkylated in 50 mM Iodoacetamide prior to boiling in loading buffer, and then separated by one-dimensional SDS-PAGE (4–12% Bis-Tris Novex mini-gel, Invitrogen) and visualized by colloidal Coomassie staining (Novex, Invitrogen). The entire protein gel lanes were excised and cut into 10 slices each. Every gel slice was subjected to in-gel digestion with trypsin overnight at 37°C. The resulting tryptic peptides were extracted by formic acid (1%) and acetonitrile (CH3CN), lyophilized in a speedvac and resuspended in 1% formic acid.
10
Protein Digestion and Separation
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Prior to protein digestion, equal amounts of protein (80 µg) from unlabelled and labelled samples were combined. Samples were reduced in 10 mM DTT and alkylated in 50 mM Iodoacetamide prior to boiling in loading buffer, and then separated by one-dimensional SDS-PAGE (4–12 % Bis–Tris Novex mini-gel, Invitrogen) and visualised by colloidal Coomassie staining (Novex, Invitrogen). The entire protein gel lanes were excised and cut into 10 slices each. Every gel slice was subjected to in-gel digestion with trypsin overnight at 37 °C. The resulting tryptic peptides were extracted by formic acid (1 %) and acetonitrile (CH3CN), lyophilized in a speedvac and resuspended in 1 % formic acid.
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