To examine the sensitivity and specificity of the established antibodies in immunoblot analysis and immunoprecipitation, the TMEPAI/V5 or C18ORF1/V5 expression plasmids were transfected into COS7 cells (5 × 106 cells/6-cm dish) using FuGENE 6 (Roche Applied Science). Thirty-six hours after the transfection, the cells were dissolved in 500 μL TNE buffer and the debris was removed by centrifugation as previously described.19 (link) For immunoprecipitation, the lysate was precleared with protein G-Sepharose beads (GE Healthcare) for 30 min at 4°C with end-over-end rotation and then incubated with anti-V5 antibody (Sigma) or anti-TMEPAI antibodies for 2 h at 4°C. The immune complexes were precipitated by incubation with protein G-Sepharose beads for 30 min at 4°C followed by three washes with TNE buffer. The immunoprecipitated proteins and aliquots of the total cell lysate were separated by SDS-PAGE, and transferred to Hybond-C Extra membranes (GE Healthcare). The membranes were probed with different primary antibodies and then incubated with HRP-conjugated secondary antibodies and chemiluminescent substrate solution (Thermo Scientific, Waltham, MA, USA). Endogenously expressed TMEPAI in HaCaT or lung cancer cell lines was also detected by immunoblot analysis using total cell lysates with/without TGF-β stimulation.
Chemiluminescent substrate solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
Chemiluminescent substrate solution is a reagent used in molecular biology and biochemistry applications. It is designed to detect and quantify the presence of specific proteins or enzymes in a sample through a chemiluminescent reaction.
Automatically generated - may contain errors
Lab products found in correlation
Las 4000 mini, by Fujifilm (2 mentions)
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Protein g sepharose bead, by GE Healthcare (1 mentions)
Anti v5 antibody, by Merck Group (1 mentions)
Hybond c extra membrane, by GE Healthcare (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Gel electrophoresis system, by Bio-Rad (1 mentions)
Hrp linked anti rabbit igg, by Cell Signaling Technology (1 mentions)
Simplyblue safe stain solution, by Thermo Fisher Scientific (1 mentions)
Pvdf immunoblot membrane, by Bio-Rad (1 mentions)
Phosphatase inhibitor cocktails 2 and 3, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
Peroxidase conjugated goat anti human igg, by Zymo Research (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Hyperfilm ecl, by Cytiva (1 mentions)
7 protocols using chemiluminescent substrate solution
1
Immunoblot and Immunoprecipitation Analysis of TMEPAI/V5 and C18ORF1/V5 Proteins
2
Western and Dot Blot Assay for NS1 Protein
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NS1 proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane by electroblotting for western immunoblot assay. For dot blot assay, purified NS1 or bacterial lysates containing NS1 protein were dotted onto nitrocellulose membrane at the desired concentration. Membranes from both assays were blocked with 5% skim milk in PBS pH 7.4 for 1 h at room temperature. After 3 times washing with PBS, the blocked membranes were incubated with anti-NS1 mAbs for 1 h at 37 °C, followed by rabbit anti-mouse immunoglobulins conjugated with HRP (Agilent, USA) for another 1 h in the dark. To visualize the protein, the membranes were incubated with chromogenic substrate solution (Diaminobenzidine; DAB and H2O2) for 5 min. When greater sensitivity was required, the membranes were incubated with chemiluminescent substrate solution (Thermo Fisher Scientific) and exposed to X-ray film in the dark.
3
Western Blot Analysis of Protein Levels
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Cells were lysed with cold RIPA buffer. After running SDS-PAGE, proteins were transferred to a PVDF membrane (Millipore) and blotted with primary antibody-diluted 4% skim milk in TBST at 4°C overnight followed by incubation with a HRP-conjugated secondary antibody. The blots were treated with chemiluminescent substrate solution (Thermo Fisher Scientific, Waltham, MA, USA) and exposed to LAS-4000 mini (Fujifilm Co., Tokyo, Japan) to reveal immunoreactive bands. Percentages from each band on densitometry compared to the control were indicated in the lower lanes in the figures. The western blot analysis was repeated 3 times.
4
Confirming Recombinant Protein Size by SDS-PAGE
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In order to confirm recombinant proteins were correctly sized, SDS PAGE was performed using a BioRad gel electrophoresis system. Samples containing recombinant EK-PSA-GZMB/TRP proteins were mixed 1:1 with 4X reducing SDS PAGE Sample Buffer and boiled for 5 minutes. Samples were then loaded and run at 150V until fully migrated. For protein staining, the gel was then washed 2-3 times with RO H2O and stained for one hour with SimplyBlue Safe Stain solution (Thermo LC6060). To de-stain samples were washed in RO H2O overnight. For Western blotting, samples were run on a non-reducing gel and were transferred to PVDF Immuno-Blot membrane (BioRad 1620177) for 1 hour at 100V and incubated for one hour in TBST containing 5% milk. To monitor removal of the DDDK peptide, an anti-DDDDK polyclonal antibody (Abcam 1162) was diluted to 1:5000 in TBST/milk and incubated with the membrane overnight at 4°C. The next day, the membrane was washed 5X with TBST and incubated with an anti-rabbit HRP-linked IgG (Cell Signaling #70762) diluted at 1:10000 in TBST/milk for one hour at 4°C. The blot was then washed 5X with TBST and incubated with chemiluminescent substrate solution (Thermo 34077) diluted according to manufacturer's instructions. The blots were then developed at specified time points.
5
Anosmin-1 and VEGF-A Signaling Pathways
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Cells were washed twice with 1 × HBSS, and then, HUVECs and PAE/KDR cells were incubated in 2% FBS EBM-2 and Ham’s F-12 without FBS for 4 h for starvation. The cells were stimulated with 7.5 nM anosmin-1 or 1 nM VEGF-A for the indicated durations and then lysed with RIPA buffer (50 mM Tris-HCl [pH 7.4], 100 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40) containing 1% phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich, St. Louis, MO, USA). After SDS-PAGE, proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA) and blotted with the phospho-specific primary Abs diluted in 5% Blocking One (Nacalai Tesque) in TBST at 4 °C overnight, followed by incubation with the HRP-conjugated secondary Ab. The blots were treated with chemiluminescent substrate solution (Thermo Fisher Scientific) and exposed to LAS-4000 Mini (Fujifilm Co., Tokyo, Japan) to visualize immunoreactive bands.
6
Quantitative Analysis of IgG Binding
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Ten µl aliquots of recombinant FcεRIαproteins (30 µg/ml in PBS) were dotted onto the centers of individual squares made on a nitrocellulose membrane (NC) and air-dried. Unoccupied sites on the NC were blocked with 0.15% casein in PBS (PBSC) for 10 min. After washing, individual patient serum was diluted 1∶4 in PBSC and incubated for 10 min. Bound IgG were detected using peroxidase-conjugated goat anti-human IgG (Zymed). The reaction was developed using a chemiluminescent substrate solution (Applied Biosystem, Bedford, MA, USA) and the signals were recorded by exposure to an X-ray film. Quantitative analysis of the spots was conducted by video densitometer using the software, Kodak Analyzer.
7
Immunoblotting Analysis of IgE Antibodies
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Protein samples were loaded on a 4% polyacrylamide stacking gel above a 12% separating gel that was run with discontinuous buffer by Laemmli's method. After electrophoresis, the gels were fixed and stained with 0.2% Coomassie brilliant blue R250. For immunoblotting, the gels were transferred electrophoretically by semidry (Bio-Rad) for 30 minutes at 0.8 mA/cm2 to nitrocellulose membranes (Millipore Bedford, MA, USA). After the transfer, the membranes were blocked in PBST (10 mM sodium phosphate, pH 7.4; 150 mM NaCl; 0.05% Tween 20) containing 5% skim milk for 2 hours at room temperature. The blots were incubated with a 1:10 dilution of patient serum pool or negative control pool overnight at 4℃, and then washed during three 20-minute periods in PBST and incubated with a 1:2,000 dilution of an anti-human IgE alkaline phosphatase conjugate (PharMingen, San Diego, CA, USA) for 2 hours at room temperature. The reaction was developed using a chemiluminescent substrate solution (Applied Biosystem, Bedford, MA, San Antonio, TX, USA). The signals were recorded by exposure to ECL Hyperfilm (Amersham Biosciences, Buckinghamshire, UK).
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