Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (MilliporeSigma) supplemented with protease inhibitor (MilliporeSigma). Protein concentrations were measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). An equal amount of protein per sample was balanced with RIPA lysis buffer, Laemmli sample buffer (Bio-Rad), and DTT. After boiling, the samples were run on an NuPAGE 3 to 8% Tris-Acetate gel (Invitrogen) and transferred to a polyvinylidene difluoride (PVDF) (0.45 μm) membrane. The membrane was blocked with 5% milk and then blotted with the primary and secondary antibodies. The primary antibodies used were RPA194 (C-1; Santa Cruz; catalog no.: sc-48385), Myc-tag clone 4A6 (Millipore; catalog no.: 05-724), PAF53 (ProteinTech; catalog no.: 16145-1-AP), and GAPDH (abcam; catalog no.: ab8245). Horseradish peroxidase–conjugated secondary antibodies were purchased from Dako. Secondary antibodies were detected using either SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) or Western Lightning Plus-ECL Enhanced Chemiluminescence Substrate (PerkinElmer) and imaged using the Bio-Rad Molecular Imager ChemiDoc XRS+ System and Image Lab Software. Protein densitometry analysis was conducted on the Image Lab Software.
Nupage 3 to 8 tris acetate gel
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The NuPAGE 3% to 8% Tris-acetate gel is a pre-cast polyacrylamide gel used for protein separation and analysis. It has a gradient of 3% to 8% acrylamide concentration, which allows for the separation of a wide range of protein molecular weights.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey blocking buffer, by LI COR (2 mentions)
Complete protease inhibitor cocktail, by Merck Group (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Molecular imager chemidoc xrs system, by Bio-Rad (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Agilent Technologies (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Gapdh, by Abcam (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Bio-Rad (1 mentions)
Rpa194 c 1, by Santa Cruz Biotechnology (1 mentions)
Western lightning plus ecl enhanced chemiluminescence substrate, by PerkinElmer (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Sample reducing agent, by Thermo Fisher Scientific (1 mentions)
Image lab 6, by Bio-Rad (1 mentions)
Pierce bca kit, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
γh2ax, by Merck Group (1 mentions)
8 protocols using nupage 3 to 8 tris acetate gel
1
Western Blot Protein Analysis Protocol
2
Quantifying Neuronal Sodium Channel Protein
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Cells were lysed as detailed in Supplementary Methods , and protein concentration was determined by bicinchoninic acid assay (Pierce BCA kit, Thermo Scientific). For detecting NaV1.2, 10-μg protein samples were boiled (2 min, 90°C) in NuPAGE LDS Sample Buffer (Invitrogen, NP0007) and Sample Reducing Agent (Invitrogen, NP0004) and run in NuPAGE 3 to 8%, Tris-Acetate Gel (Invitrogen, EA0378BOX). Details of blotting, antibody incubation and signal detection are available in Supplementary Material, Table S5 . Relative NaV1.2 protein levels (ratio between NaV1.2 and TUBB3 band density) were quantified using Image Lab 6.1 software (BioRad).
3
Investigating Ack1 and WASP Phosphorylation
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Huh7 cells were treated with 100 nM 10M-D42AN for 0 to 48 h and with EGF (Invitrogen) (0–100 ng/ml) for 0 to 3 h. To observe the phosphorylation levels of Ack1 and WASP, HepG2 cells were transfected with T7-Ack1-His-pcDNA using Lipofectamine 2000 and then cultured for 48 h. These cells were lysed with RIPA buffer containing a protease inhibitor cocktail and phosphatase inhibitor cocktail. Lysates were centrifuged at 4 °C and 13,200 rpm for 15 min, and the supernatant was removed. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Thermo). Equivalent amounts of protein were separated in NuPAGE 4% to 12% Bis–Tris gel (Thermo) or NuPAGE 3% to 8% Tris-acetate gel (Thermo) followed by incubation with primary antibodies against Ack1, EGFR, phosphorylated-EGFR Y845, pN-WASP (Santa Cruz), pAck1, GAPDH (Abcam, CST), Chk1, pChk1, phosphorylated-EGFR Y1045, Y1068, N-WASP (CST), and γH2AX (Merck) and with HRP-conjugated antibodies against FLAG, T7 (Novagen), and GST (Santa Cruz). HRP-conjugated secondary antibody against rabbit or mouse (Abcam, CST) was then applied. The blots were developed using enhanced chemiluminescence reagents (Nacalai Tesque, Cytiva).
4
Electrophoresis of Recombinant Spidroins
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Recombinant spidroin samples were electrophoresed on a Mini-Protean TGX 4 to 20% denaturing gel (Bio-Rad). Samples extracted from spider silk glands were run on a NuPAGE 3 to 8% tris-acetate gel (Thermo Fisher Scientific) under denaturing conditions. Gels were stained with 0.1% Coomassie brilliant blue in 40% ethanol and 10% acetic acid and destained with Milli-Q water.
5
Western Blot Analysis of TA Muscle Proteins
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TA muscles were homogenized in modified RIPA buffer (50 mM Tris [pH 8], 150 mM NaCl, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 10% SDS, and 1× cOmplete protease inhibitor cocktail [Merck, Feltham, UK]) using a Precellys 24 tissue homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France) (four cycles at 5,000 rpm for 30 s). Twenty micrograms of protein was mixed with NuPAGE sample reducing agent and NuPAGE LDS sample buffer and run on a NuPAGE 3% to 8% Tris-acetate gel (all Thermo Fisher Scientific, Abingdon, UK). Protein was electrotransferred onto a 0.45 μm polyvinylidene difluoride (PVDF) membrane for 1 h at 30 V, followed by 1 h at 100 V, and blocked with Odyssey blocking buffer (LI-COR Biosciences, Cambridge, UK) for 1 h at room temperature. Membranes were incubated with primary antibodies (Table S3 ) overnight in Odyssey blocking buffer supplemented with 0.1% Tween 20 at 4°C. Membranes were washed three times with PBST and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Table S3 ) in Odyssey blocking buffer supplemented with 0.1% Tween 20 at room temperature for 1 h. Chemiluminescent signal was detected using Clarity Western ECL substrate (Bio-Rad, Watford, UK).
6
Muscle Protein Extraction and Western Blot
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TA muscles were homogenized in modified RIPA buffer (50 mM Tris pH8, 150 mM NaCl, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 10% SDS and 1× cOmplete protease inhibitor cocktail (Merck, Feltham, UK)) using a Precellys 24 Tissue Homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France) (4 cycles at 5,000 rpm for 30 seconds). 20 µg of protein was mixed with NuPAGE sample reducing agent and NuPAGE LDS sample buffer and run on NuPAGE 3 to 8% Tris-Acetate gel (all Thermo Fisher Scientific, Abingdon, UK).
Protein was electrotransferred onto a 0.45 µm polyvinylidene difluoride (PVDF) membrane for 1 hour at 30 V, followed by 1 hour at 100 V and blocked with Odyssey blocking buffer (LI-COR Biosciences Ltd, Cambridge, UK) for 1 hour at room temperature. Membranes were incubated with primary antibodies (Table S2) overnight in Odyssey blocking buffer supplemented with 0.1% Tween-20 at 4°C. Membranes were washed 3 times with PBST and then incubated with HRP-conjugated secondary antibodies (Table S2) in Odyssey blocking buffer supplemented with 0.1% Tween-20 at room temperature for 1 hour. Chemiluminescent signal was detected using Clarity Western ECL substrate (Bio-Rad, Watford, UK).
Protein was electrotransferred onto a 0.45 µm polyvinylidene difluoride (PVDF) membrane for 1 hour at 30 V, followed by 1 hour at 100 V and blocked with Odyssey blocking buffer (LI-COR Biosciences Ltd, Cambridge, UK) for 1 hour at room temperature. Membranes were incubated with primary antibodies (Table S2) overnight in Odyssey blocking buffer supplemented with 0.1% Tween-20 at 4°C. Membranes were washed 3 times with PBST and then incubated with HRP-conjugated secondary antibodies (Table S2) in Odyssey blocking buffer supplemented with 0.1% Tween-20 at room temperature for 1 hour. Chemiluminescent signal was detected using Clarity Western ECL substrate (Bio-Rad, Watford, UK).
7
Yeast Viroporin Disulfide Mapping
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Two OD600 (optical density at 600 nm) units of yeast cells expressing WT 1a or 1a viroporin mutants were spheroplasted and then treated with 1 M sorbitol containing 10 mM N-ethylmaleimide (MilliporeSigma) for 10 min at room temperature. Spheroplasts were then lysed in 1 ml of 10% trichloroacetic acid (TCA), and total protein was collected by centrifugation for 5 min at 4°C at 15,000g. Protein pellets were washed with acetone twice. Dried pellets were suspended in 200 μl of 2× SDS loading buffer (Bio-Rad) containing 10 mM N-ethylmaleimide and vortexed for 10 min at 40°C. SDS-PAGE samples were divided into two, treated with or without DTT (final 100 mM), and vortexed for 10 min at 40°C. SDS-PAGE was performed using NuPAGE 3 to 8% tris-acetate gels (Thermo Fisher Scientific). After SDS-PAGE, the gels were soaked in a DTT-containing, alkaline transfer buffer (25 mM tris base, 190 mM glycine, 10 mM DTT) for 10 min at room temperature to break disulfide bonds of proteins and facilitate protein transfer to polyvinylidene difluoride (PVDF) membranes.
8
Western Blot Analysis of Tight Junction Proteins
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Frozen tissue samples (15 mg) were crushed, and protein and RNA was immediately extracted using the NucleoSpinR RNA/protein purification kit (Machery-Nalgel EURL, Hoerdt, France), according to manufacturer's instructions. Proteins (40 μg) were separated on Nupage 3 to 8% Tris acetate gels (Thermo Fisher Scientific, Villebon sur Yvette, France) and transferred onto polyvinylidene difluoride membranes using the Pierce Power Blotter (Thermo Scientific Pierce, Villebon sur Yvette, France). Membranes were incubated with primary antibodies overnight 4°C: a rabbit monoclonal anti-β-actin antibody (1:2000, #4970 Cell Signaling Technology, Danvers, MA), a rabbit monoclonal anti-occludin antibody (1:2000, ab167161, Abcam, Cambridge, UK), a rabbit polyclonal antizonula occludens-1 (ZO-1) antibody (1:250, 61-7300, Life Technologies, Villebon sur Yvette, France), and a mouse monoclonal anti-Muc2 (1:200, ab118964, Abcam). Bound antibodies were detected using the WesternBreeze Immunodetecting Chemiluminescent System (Invitrogen, Carlsbad, CA). The optical density of the bands was visualized with the Image System (ImageMaster VDS-CL, Amersham Biosciences, Little Chalfont, UK) and densitometrically analyzed using the Quantity One image analysis software (Bio-Rad, Hercules, CA).
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