Worms of each genotype (2000~3000 individuals of L4 stage worms after pathogen exposure) were collected and washed with M9 buffer and boiled in home-made sample buffer. Samples were boiled, spun down, and supernatants were flash frozen in dry ice and then heated at 80°C for 10 min. Proteins were resolved on a 12% SDS-polyacrylamid gel by electrophoresis, transferred to a PVDF membrane, and incubated with rabbit anti-phospho-p38 MAPK (Cell Signaling, #9211) at a 1:2,000 dilution, mouse anti-tubulin (Sigma, #T9026) at a 1:4,000 dilution, or anti-p38 MAPK at a 1:1000 dilution (obtained from Dr. Read Pukkila-Worley at UMass Medical School). Following washing, the blots were incubated with 1:2,000 secondary HRP conjugated anti-mouse (for anti-tubulin), or anti-rabbit (for anti-phospho-p38 and anti-p38 MAPK) antibody for 1 hour and subsequently washed 3 times for 3 min intervals. Blots were developed using SuperSignal™ West Atto Ultimate Sensitivity Substrate (ThermoFisher, A38555) and visualized using a ChemiDoc™ MP Imaging System (BIO-RAD). Image lab (software) was used to quantify the intensity of the immunoblot bands.
Supersignal west atto ultimate sensitivity substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States
SuperSignal™ West Atto Ultimate Sensitivity Substrate is a chemiluminescent substrate designed for high-sensitivity detection of target proteins in Western blot applications. The substrate generates a stable, long-lasting light signal that can be detected using appropriate imaging equipment.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Thermo Fisher Scientific (3 mentions)
Ibright fl1500 imaging system, by Thermo Fisher Scientific (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Protein quantification kit, by Beyotime (2 mentions)
Ripa radioimmunoprecipitation assay buffer, by Thermo Fisher Scientific (2 mentions)
Mini protean tgx stain free protein gel, by Bio-Rad (2 mentions)
Pierce clear milk blocking buffer, by Thermo Fisher Scientific (2 mentions)
Mouse anti human igg ch3 domain secondary antibody, by Thermo Fisher Scientific (2 mentions)
Ibright analysis software version 5, by Thermo Fisher Scientific (2 mentions)
Tbs tween, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase hrp conjugated horse anti mouse 7075, by Cell Signaling Technology (2 mentions)
Image lab software package, by Bio-Rad (2 mentions)
Luminata forte western hrp substrate, by Merck Group (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Rabbit anti phospho p38 mapk, by Cell Signaling Technology (1 mentions)
Anti p38 mapk, by Merck Group (1 mentions)
19 protocols using supersignal west atto ultimate sensitivity substrate
1
Phosphorylation of p38 MAPK in C. elegans
2
Retinal Ganglion Cell Purification and Analysis
3
Glioblastoma Protein Expression Analysis
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Glioblastoma cells were lysed in lysis buffer with protease inhibitor cocktails (Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations were determined by Bradford assay (Bio-Rad, Hercules, CA, USA). For Western blot analysis, equal amounts of protein were separated on 8–15% sodium dodecyl sulphate (SDS)-polyacrylamide gels and transferred to PVDF membranes. Blots were blocked for 1 h at room temperature with blocking buffer. Membranes were incubated overnight in a cold chamber with specific antibodies. After being washed with Tris-buffered saline (TBS), membranes were incubated with a horseradish-peroxidase-labeled secondary antibody (Abcam) and visualized using a Supersignal west atto ultimate sensitivity substrate (Thermo Scientific, Waltham, MA, USA; A38555).
4
Western Blot Analysis of ADH4 Protein
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ADH4 protein expression of the resected tissues was examined using western blotting. The tissues were first treated using a lysis solution (RIPA, Radio-Immunoprecipitation Assay buffer; Thermo Fisher Scientific, USA), which contained 1% (v/v) protease inhibitors (cat. no. P8340; Merck KGaA) to extract protein samples. The protein concentration was measured and normalized using Protein Quantification kit (BCA Assay; Beyotime, China). Western blot analysis was then performed as previously described (18 (link)). The membranes were blocked in Tris-Buffer Saline Tween 20 (1×TBST) containing 5% non-fat milk at room temperature for 45 min. Then, membranes would be incubated with anti-ADH4 primary antibody (cat. no. Ab137077; 1:1,000 dilution; Abcam) overnight at 4°C and HRP-conjugated goat anti-rabbit secondary antibody (cat. no. Ab6721; 1:1,000 dilution; Abcam) for 1 h at room temperature. Enhanced chemiluminescence reagent (SuperSignal™ West Atto Ultimate Sensitivity Substrate; Thermo Fisher Scientific, Inc.) was used for immunodetection. The level of actin protein expression was measured as an internal standard (anti-β actin antibody; cat. no. Ab8227; 1:1,000 dilution; Abcam). The density of protein band was quantitatively measured using Image J software (v1.52a, USA).
5
Characterization of Extracellular Vesicle Markers
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For membrane markers, 40
μL of sEVs
(1 × 1010 particles/mL) and for intraluminal and negative
markers, 1 × 1010 particles in 40 μL were spotted
on nitrocellulose membranes (0.45 μm; Bio-Rad) and incubated
at RT for 1 h in blocking buffer (3% milk in TBS-T). Mouse anti-human
CD63 (BioLegend #353013), CD81 (BioLegend #349520), CD9 (BioLegend
#312102), and Calnexin (GeneTex #GTX629976-S) antibodies at 0.5 μg/mL
and Alix (Cell Signalling Technology #2171S) at 0.2 μg/mL in
blocking buffer were added to separate membranes and incubated overnight
at 4 °C. Staining was performed with an HRP-conjugated goat anti-mouse
IgG antibody (1:10,000 dilution in blocking buffer; BioLegend #405306)
for 1 h at RT. A chemiluminescence signal was detected using a SuperSignal
West Atto Ultimate Sensitivity substrate (Thermo Fisher), imaged on
iBright FL1000 (Invitrogen) or developed on a CL-Exposure film (Thermo
Fisher).
μL of sEVs
(1 × 1010 particles/mL) and for intraluminal and negative
markers, 1 × 1010 particles in 40 μL were spotted
on nitrocellulose membranes (0.45 μm; Bio-Rad) and incubated
at RT for 1 h in blocking buffer (3% milk in TBS-T). Mouse anti-human
CD63 (BioLegend #353013), CD81 (BioLegend #349520), CD9 (BioLegend
#312102), and Calnexin (GeneTex #GTX629976-S) antibodies at 0.5 μg/mL
and Alix (Cell Signalling Technology #2171S) at 0.2 μg/mL in
blocking buffer were added to separate membranes and incubated overnight
at 4 °C. Staining was performed with an HRP-conjugated goat anti-mouse
IgG antibody (1:10,000 dilution in blocking buffer; BioLegend #405306)
for 1 h at RT. A chemiluminescence signal was detected using a SuperSignal
West Atto Ultimate Sensitivity substrate (Thermo Fisher), imaged on
iBright FL1000 (Invitrogen) or developed on a CL-Exposure film (Thermo
Fisher).
6
Western Blot Analysis of BMAL1 and p-BMAL1
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U2-OS and U2-OS-Luc cells were washed with PBS and then lysed using RIPA buffer (Cell Signaling, Stockholm, Sweden) with a protease inhibitor cocktail (Roche, Stockholm, Sweden). The collected cell lysate was used to determine the protein concentration with a BCA Kit (Thermo Fisher Scientific). For each sample, 20 µg of protein was resolved in a 4–15% SDS-PAGE gel (Bio-Rad, Solna, Sweden) along with a protein ladder (Bio-Rad, Solna, Sweden) and transferred onto PVDF membranes. The transferred membranes were blocked in 5% milk in TBST (25 mM Tris-HCl, pH 7.4, 125 mM NaCl, 0.05% Tween 20) for 1 h and incubated with the primary antibodies at 4 °C overnight. After the membrane was washed thrice with TBST for 7 min, it was incubated with horseradish–peroxidase-coupled isotype-specific secondary antibodies (Dako) for 1 h at room temperature. Detection was performed by SuperSignal™ West Atto Ultimate Sensitivity Substrate according to the manufacturer’s instructions (Thermo Fisher Scientific). Primary antibodies for BMAL1 and p-BMAL1 were from Thermo Fisher Scientific.
7
SDS-PAGE Analysis of Fusion Proteins
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To evaluate the correct molecular weight of the different fusion proteins, the supernatant of transfected HEK293T cells was run with β-mercaptoethanol (Sigma-Aldrich, #63689) on a 4-20% Mini-Protean® TGX Stain-Free™ Protein Gel (BioRad, #4568093). After transfer to PVDF membranes (ThermoFisher, #88518), the membranes were blocked with 1x Pierce™ Clear Milk Blocking Buffer (ThermoFisher, #37587) and incubated overnight with Mouse anti-Human IgG (CH3 domain) Secondary Antibody (Invitrogen, #MA5-16557). After washing with TBS-Tween (ThermoFisher, #28360), the membranes were incubated for 45 min with Anti-mouse IgG HRP-conjugated (R&D systems, #HAF007) and developed using SuperSignal™ West Atto Ultimate Sensitivity Substrate (ThermoFisher, #A38555). Images were obtained using the iBright FL1500 Imaging System (Invitrogen, #A44241) and analyzed with iBright Analysis Software Version 5.1.0 (Invitrogen).
8
Identification of circCUX1-Binding Proteins
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The biotin-labeled circCUX1 probe was mixed with total cell protein and incubated for 24 h at 4 °C to make circCUX1 and protein form a circCUX1-protein complex, and then the magnetic beads (MEGA clear Kit, Ambion) were used to adsorb the complex. The protein in the capture complex was analyzed by Western blotting, using sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE) electrophoresis. The proteins were transferred on polyvinylidene fluoride (PVDF) membrane. The membrane was incubated with 5% non-fat milk for 1 h at 37 °C and incubated with a primary antibody METTL3 (diluted 1:1000, Abcam) overnight at 4 °C. After washed with PBS, the membrane was incubated with the secondary antibody for 2 h at room temperature. A SuperSignal West Atto Ultimate Sensitivity Substrate (cat no. A38555, ThermoFisher, USA) was used to protein blot detection in the gel imaging system.
9
SDS-PAGE Analysis of Fusion Proteins
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To evaluate the correct molecular weight of the different fusion proteins, the supernatant of transfected HEK293T cells was run with β-mercaptoethanol (Sigma-Aldrich, #63689) on a 4-20% Mini-Protean® TGX Stain-Free™ Protein Gel (BioRad, #4568093). After transfer to PVDF membranes (ThermoFisher, #88518), the membranes were blocked with 1x Pierce™ Clear Milk Blocking Buffer (ThermoFisher, #37587) and incubated overnight with Mouse anti-Human IgG (CH3 domain) Secondary Antibody (Invitrogen, #MA5-16557). After washing with TBS-Tween (ThermoFisher, #28360), the membranes were incubated for 45 min with Anti-mouse IgG HRP-conjugated (R&D systems, #HAF007) and developed using SuperSignal™ West Atto Ultimate Sensitivity Substrate (ThermoFisher, #A38555). Images were obtained using the iBright FL1500 Imaging System (Invitrogen, #A44241) and analyzed with iBright Analysis Software Version 5.1.0 (Invitrogen).
10
Western Blot Analysis of Cell Signaling
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Cells were lysed in RIPA lysis buffer (Epizyme, PC101) supplemented with a protease/phosphatase inhibitor cocktail. Proteins were quantified by BCA Protein Assay Kit (Epizyme, ZJ101), supplemented with the loading buffer, and placed in a metal bath at 95 °C for 10 min. After being separated by SDS–polyacrylamide gel electrophoresis, proteins were transferred to PVDF membranes and blocked in PBS containing 5% BSA and 0.1% Tween20. Then, membranes were incubated with primary antibodies (DR6 (1:100, rabbit, bs-7678R, Bioss), Cldn-5 (1:1000, rabbit, AF5216, Affinity), Glut-1 (1:1000, rabbit, ab115730, Abcam), active β-catenin (1:1000, rabbit, 8814, CST), total β-catenin (1:1000, rabbit, 8480, CST), Phospho-JNK (1:1000, rabbit, AP1337, Abclonal), JNK (1:1000, rabbit, A4867, Abclonal) and HRP-Conjugated GAPDH (1:10,000, HRP-60004, Proteintech)) overnight at 4 °C, followed by corresponding secondary antibodies, and detected with SuperSignal West Atto Ultimate Sensitivity Substrate (Thermo Fisher Scientific, A38555) by ChemiDoc MP Imaging System (Bio-Rad, USA). Fiji software was used to quantify band densities. The quantification of phosphorylated proteins was normalised to their total proteins. GAPDH was used as the housekeeping protein, and the data were presented as fold change to the control group.
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