Capsid thermal stability assays were performed as described before [37 (link)]. Briefly, per reaction 1x1010 viral capsids of a vector preparation were diluted in PBS, incubated at indicated temperatures for 15 min and then further diluted in PBS. The temperature gradient (65 to 85°C) was generated by a LightCycler® 96 System (Roche Life Science) with the following program: 2 cycles (10 sec at 37°C; 900 sec at temperature gradient; 10 sec at 37°C), 1 cycle (30 sec at 37°C). Samples were transferred to a nitrocellulose membrane using a vacuum blotter, blocked (5% milk in TBS-T) and incubated with hybridoma cell supernatant containing anti-AAV2 specific antibody A20 (recognizing intact capsids) (PROGEN, 1:5 dilution). Anti-mouse IgG HRP-conjugated antibody was used as secondary antibody (Cayman Chemical—Biomol, Hamburg, Germany; 1:2,000 dilution) and signals were detected using SuperSignal™ West Pico Chemiluminescent Substrate or SuperSignal™ West Femto Maximum Sensitivity (ThermoFisher Scientific, Waltham, MA) in combination with a Fusion FX Vilber Lourmat system (Peqlab).
Supersignal west femto maximum sensitivity
Manufactured by Thermo Fisher Scientific
Sourced in France
The SuperSignal™ West Femto Maximum Sensitivity is a chemiluminescent substrate used for the detection of low-abundance proteins in Western blotting applications. It provides a sensitive and reliable method for visualizing target proteins with high signal-to-noise ratios.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Anti mouse igg hrp conjugated antibody, by Cayman Chemical (2 mentions)
Sc 8334, by Santa Cruz Biotechnology (2 mentions)
Ms 1295 p, by Merck Group (2 mentions)
Stat1a, by Santa Cruz Biotechnology (2 mentions)
Immobilon western chemoluminescent hrp, by Merck Group (2 mentions)
Gapdh, by Merck Group (2 mentions)
Flag tag (flag), by Merck Group (2 mentions)
Hrp conjugated secondary antibody, by GE Healthcare (2 mentions)
Mini protean tgx gel, by Bio-Rad (2 mentions)
Lightcycler 96 system, by Roche (1 mentions)
Extraavidin peroxidase, by Merck Group (1 mentions)
Anti mouse igg peroxidase, by Merck Group (1 mentions)
Chemidoc mp detection system, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Mouse α flag m2, by Merck Group (1 mentions)
Mouse α dnak, by Stressgen (1 mentions)
Clarity western ecl, by Bio-Rad (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
10 protocols using supersignal west femto maximum sensitivity
1
Capsid thermal stability assay
2
Western Blot Analysis of Viral Proteins
3
Quantitative Protein Analysis by Western Blot
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Western blots were performed on cell extracts from HT29. Protein concentrations of extracts were determined by Uptima CooAssay Max Protein Assay Kit (Interchim, UP87542A) to ensure equal loading. Proteins were separated by electrophoresis on 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for HSPB1 detection and 10% SDS-PAGE for HSPA1A detection. HSPB1 (StressMarq, SPR-105B) and HSPA1A (StressMarq, SPR-103B) human recombinant proteins were used as standard controls. Separated proteins were transferred onto nitrocellulose membranes using a Bio-Rad Trans-Blot Turbo Transfer System. HSPB1 was detected using a monoclonal HSPB1 antibody (StressMarq, SMC-161A) at 1:5000 dilution as the primary antibody and anti-mouse IgG–peroxidase (Sigma-Aldrich, A5278) at 1:2000 as the secondary antibody. HSPA1A was detected by HSPA1A-DEG-EI (in house) diluted at 1:2000 as the primary antibody and ExtraAvidin®-Peroxidase (Sigma-Aldrich, E2886) diluted 1:5000 as secondary antibody. Membranes were washed with TBS-Tween 20 and then incubated for 5 min in SuperSignal West Femto Maximum Sensitivity (Thermo Scientific) substrate. Chemiluminescence was detected using the ChemiDoc MP detection system (Bio-Rad, Hercules, CA, USA).
4
Western Blot Analysis of Bacterial Proteins
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Protein fractions were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane, and blocked in Tris-buffered saline with 0.1% (w/v) Tween (TBST) containing 5% skim milk. Membranes were then probed using the following primary antibodies: mouse monoclonal anti-Tir (1:2000) for full-length and C-terminal fragments, rat polyclonal anti-Tir from C. rodentium (1:2000) [49 (link)] for N-terminal fragments, rat polyclonal anti-NleA from EHEC (1:2000) [50 (link)], mouse α-DnaK (Stressgen, 1:5000), mouse α-FLAG M2 (Sigma, 1:5000), mouse α-His6 (GE Healthcare, 1:3000), or goat α-GAPDH (R&D Systems Inc., 1:5000). The blots were then developed using the following secondary antibodies: goat anti-mouse (1:5000, Jackson), goat anti-rat (1:2000, EMD Millipore), or donkey α-goat (Santa Cruz Biotechnology, 1:5000) conjugated to horseradish peroxidase, and imaged using the Clarity Western ECL (BioRad) or SuperSignal West Femto Maximum Sensitivity (ThermoFisher) substrates and a ChemiDoc XRS+ (BioRad).
5
Western Blot Analysis of Viral Proteins
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Whole cell extracts were separated on Mini Protean TGX gels and transferred onto nitrocellulose membrane (BioRad®). SINV-specific ascites fluid (ATCC-VR-1248AF) and antibodies specific to actin (Thermo Fisher®, MS-1295-P), GAPDH (Sigma®, G9545), Flag (Sigma®, F1804), NS1 (CTAD, Icahn School of Medicine at Mount Sinai), STAT1a (Santa Cruz, c-111), and RIG-I (Generously provided by Dr. Adolfo Garcia-Sastre, Icahn School of Medicine at Mount Sinai, NY) were used at a dilution of 1:1000 in 5% milk. Russian spring-summer encephalitis virus ascites fluid (ATCC- V558701562) was used for the detection of Langat virus at a dilution of 1:500 in 5% milk. Antibodies specific to GFP (Santa Cruz, sc-8334) and RRV-specific ascites fluid (ATCC-VR-1246AF) were used at a dilution of 1:200 in 5% milk. The SINV nsP4 antibody was a kind gift from Dr. Richard W. Hardy (Indiana University, Bloomington, IN) and was used at a 1:5,000 dilution in 5% milk in 25 mM Tris-HCl [pH 7.4], 137 mM NaCl, 2.7 mM KCl. and 0.1% Tween20. Mouse and rabbit HRP-conjugated secondary antibodies (GE Healthcare®) were used at a 1:2000 dilution in 5% milk. For antibody detection, Immobilon Western Chemoluminescent HRP (Millipore®) and SuperSignal™ West Femto Maximum Sensitivity (Thermo Fisher®) substrates were used according to the manufacture’s instructions.
6
AAV2 Capsid Composition Analysis by Western Blot
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Capsid composition was analysed by Western Blot. Iodixanol-purified viral vector particles were concentrated using the StrataClean Resin (Agilent Technologies, Ratingen, Germany) according to manufacturer’s instructions, separated on a 8% SDS-PAGE gel (5x1010 capsids per lane) and subjected to standard Western blotting procedure using the following primary antibodies: hybridoma cell supernatant containing AAV2 specific antibody A69 (recognizing VP1/VP2 proteins [36 (link)], 1:10 dilution) (PROGEN, Heidelberg, Germany). Anti-mouse IgG HRP-conjugated antibody was used as secondary antibody (Cayman Chemical—Biomol, Hamburg, Germany; 1:2,000 dilution) and signals were detected using the SuperSignal™ West Pico Chemiluminescent Substrate or SuperSignal™ West Femto Maximum Sensitivity (ThermoFisher Scientific, Waltham, MA) in combination with a Fusion FX Vilber Lourmat system (Peqlab).
7
Immunoblotting for Protein Quantification
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The protein levels in cells were determined by immunoblotting as previously mentioned23 (link). Briefly, cells were washed with ice cold PBS and lysed in MPER lysis buffer (Thermo Fischer Scientific) containing protease inhibitors cocktail (Thermo Fischer Scientific) for 15 min on ice. The cell debris was removed by centrifuge at 10000 × g for 15 min at 4 °C. The protein levels in clear cell lysate were measured by Bradford assay (VWR Life Science). Total protein (40 μg) was resolved by SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad). The membrane was blocked in 5% skim milk for 45 min at room temperature followed by probing with 1:1000 diluted primary antibodies (1:250 for ORP5) overnight at 4 °C. After three washes with TBST (50 mM Tris-Cl, 150 mM NaCl, and 0.1% Tween 20; pH7.6), membrane was incubated with respective HRP-conjugated secondary antibodies (1:1000) for 2 h at room temperature and washed three times. The luminescent signal was developed either using Super Signal West Femto Maximum Sensitivity or Dura Extended Duration substrate (Thermo Fischer Scientific) and images were captured using Gel Doc system (Bio-Rad). Image Lab software (Bio-Rad) was used for densitometry of bands.
8
Western Blot Protein Detection
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In brief, proteins were size fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred onto nitrocellulose or PVDF membrane. Saturation and blotting were realized by using SNAP i.d™ Protein Detection System (Millipore, France). The detection of proteins was performed using ECL™(Amersham Biosciences, France) and/or SuperSignal west femto Maximum Sensitivity (Thermo Scientific, France) chemilumenscence reagents. The detection of proteins was performed using the FusionX7 Imager (Fisher Scientific, France).
9
Melatonin Regulation of Schwann Cell Signaling
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Schwann cells treated with melatonin were lysed with 10X SDS sample buffer [62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol and 100 mM DTT]. Protein concentration was determined using the Quick Start™ Bradford Protein Assay kit (Bio-Rad Laboratories, Inc.). Protein samples (25 µg) were applied to 10% gels and separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% BSA for 1 h at room temperature and then incubated with primary antibodies against MT1, MT2, GDNF, PKC, Ras, B-raf, p-B-Raf, C-raf, p-C-Raf, ERK1/2, p-ERK1/2, p39, p-p38, SAPK-JNK, p-SAPK-JNK and α-tubulin at 4˚C overnight. All primary antibodies were prepared in 1:500 dilutions in blocking buffer. The blots were then incubated with horseradish peroxidase-conjugated anti-rabbit/mouse secondary antibodies at 1:10,000 dilution for 1 h at room temperature. The blots were subsequently developed with SuperSignal™ West Femto Maximum Sensitivity (Thermo Fisher Scientific, Inc.) substrate and visualised using ChemiDoc XRS+ system (Bio-Rad Laboratories, Inc.). Quantitative blot data were analysed and calculated using Image Lab Software (version 4.0; Bio-Rad Laboratories, Inc.).
10
Immunoblotting of Protein Lysates
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Protein pellets were lysed in 1 × Nonidet P-40 lysis buffer (1% Nonidet P-40, 50 mM Tris HCl, 150 mM NaCl (pH 7.5), EGTA, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 100 mM NaF, and 1 mM PMSF) incubated on ice for 20 minutes with intermittent vortexing and centrifuged at 13000 rpm for 15 minutes at 4°C. The supernatants containing the proteins were collected and quantified using BCA reagents (G Biosciences). Lysates were mixed with 6× denaturing dye and the proteins were resolved using SDS-PAGE and transferred to PVDF membranes. The membranes were blocked in 5% BSA dissolved in 1× TBST before the addition of primary antibodies. Primary antibodies against the proteins of interest were diluted in the blocking buffer, added to the membrane and incubated overnight at 4°C. Later, the membranes were washed in 1× TBST, secondary antibodies conjugated with HRP were added and the blots were developed on a Bio-Rad Chemidoc MP system using SuperSignal West Pico PLUS (Thermo Fisher) and SuperSignal West Femto Maximum Sensitivity (Thermo Fisher) chemiluminescent substrate kits.
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