To examine proteins on MDCK and HEK293 cell surface, transfected cells expressing corin were treated with 0.25% (w/v) trypsin and 0.02% EDTA (w/v) (Gibco, 25200) at 37°C for 30 s. DMEM with 10% FBS was added to neutralize trypsin activity. After washing with PBS, the cells were lysed in a buffer containing 1% (v/v) Triton X-100, 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, and a protease inhibitor mixture (1:100, Roche Applied Science, 04693116001). Proteins were separated by SDS-PAGE under reducing conditions with 2.5% (v/v) β-mercaptoethanol in the Laemmli buffer (Bio-Rad) and transferred onto polyvinylidene difluoride membranes (Thermo Fisher) in an apparatus (Bio-Rad Trans-Blot, 300 mA) with 25 mM Tris base, 190 mM glycine, and 20% (v/v) methanol at 4°C for 60 min. Western blotting was done using a horseradish peroxidase (HRP)-conjugated anti-V5 antibody (1:5000, Thermo Fisher, R96125). After incubation with a chemiluminescent substrate (EZ-ECL) (Biological Industries, K-12045), western blots were exposed to X-ray films, which were analyzed by a scanner (CanoScan LiDE 110, Canon).
Hrp conjugated anti v5 antibody
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The HRP-conjugated anti-V5 antibody is a laboratory reagent used for the detection and visualization of proteins tagged with the V5 epitope. The antibody is conjugated with horseradish peroxidase (HRP), which enables colorimetric or chemiluminescent detection when the antibody binds to the V5 tag. This antibody can be used in various immunoassay techniques, such as Western blotting, ELISA, and immunohistochemistry, to identify and quantify V5-tagged proteins.
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Lab products found in correlation
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (2 mentions)
Protease inhibitor mixture, by Roche (2 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (1 mentions)
Ez ecl, by Sartorius (1 mentions)
Canoscan lide110, by Canon (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Trans blot, by Bio-Rad (1 mentions)
Imager 600, by Cytiva (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Mouse monoclonal anti gfp antibody, by Roche (1 mentions)
Peroxidase conjugated goat anti rabbit igg, by Dianova (1 mentions)
Goat anti rabbit igg coupled to alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Rabbit anti lc3, by Abcepta (1 mentions)
Rabbit anti mnsod, by Merck Group (1 mentions)
Goat anti mouse igg, by Dianova (1 mentions)
Anti mouse hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Anti flag m2 antibody, by Merck Group (1 mentions)
Mg132, by Merck Group (1 mentions)
5 protocols using hrp conjugated anti v5 antibody
1
Detecting Cell Surface Proteins by Western Blot
2
TMPRSS11A Protein Expression Analysis
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HEK293, EC9706, 16HBE, and A549 cells transfected with plasmids expressing TMPRSS11A proteins or a control vector were cultured, as described above. When the cell culture reached confluence, the conditioned medium was collected. TMPRSS11A proteins in the conditioned medium were immunoprecipitated using the anti-V5 antibody. The cells were lysed in a buffer containing 1% (v/v) Triton X-100, 50 mm Tris-HCl (pH 8.0), 150 mm NaCl, and a protease inhibitor mixture (1:100, Roche Applied Science, 04693116001). The proteins from the conditioned medium and cell lysates were mixed with a Laemmli sample buffer (Bio-Rad) with (reducing) or without (nonreducing) β-mercaptoethanol (2.5% v/v), and analyzed by SDS-PAGE and Western blotting using a horseradish peroxidase (HRP)-conjugated anti-V5 antibody (1:5000, Thermo Fisher, R96125). After incubation with a solution with an enhanced chemiluminescent substrate (NcmECL Ultra) (NCM Biotech, P10100), Western blots were exposed to a chemiluminescent imager (Amersham Biosciences Imager 600).
3
Surface Protein Isolation from HEK293 Cells
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To examine proteins on HEK293 cell surface, transfected cells expressing TMPRSS11A and control cells expressing corin were treated with 0.25% (w/v) trypsin and 0.02% EDTA (w/v) (Gibco, 25200) at 37 °C for 30 s. Dulbecco's modified Eagle's medium with 10% FBS was added to neutralize trypsin activity. After washing with PBS, the cells were lysed in the lysis buffer as described above. Proteins were separated by SDS-PAGE under reducing conditions with 2.5% (v/v) β-mercaptoethanol in the Laemmli buffer. Western blotting was done using an HRP-conjugated anti-V5 antibody (1:5000, Thermo Fisher, R96125), as described above.
4
Immunoblotting antibodies for mitochondrial proteins
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Rabbit anti-human GCDH antibody was kindly provided by Dr. S. I. Goodman (University of Colorado Health Sciences Center, Denver). The polyclonal mouse anti-human DLST and rabbit anti-human ETFA antibodies were purchased from Sigma (Munich, Germany), rabbit anti-human ETFB from Abcam (Cambridge, UK), and rabbit anti-LC3 from Abgent (San Diego, USA). The monoclonal mouse anti-GFP antibody was obtained from Roche (Mannheim, Germany) and rabbit anti-MnSOD from Millipore (Billerica, USA). Peroxidase-conjugated goat anti-rabbit IgG and goat anti-mouse IgG was from Dianova (Hamburg, Germany). HRP-conjugated anti-V5 antibody, monkey anti-mouse IgG coupled to Alexa Fluor 488 and goat anti-rabbit IgG coupled to Alexa Fluor 546 were from Invitrogen (Karlsruhe, Germany).
5
Immunoprecipitation and Immunoblotting of FLAG-Tagged Proteins
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COS-7 cells were transfected with expression plasmids overnight and incubated with or without the proteasome inhibitor MG132 (Merck, Kenilworth, NJ, USA) at 20 μmol·L−1 for 2 h. The cells were then lysed in NP-40 lysis buffer (150 mmol·L−1 NaCl, 1% NP-40, 50 mmol·L−1 Tris-HCl; pH 8.0) supplemented with aprotinin and phenylmethylsulfonyl fluoride (PMSF). The lysate was immunoprecipitated with an anti-FLAG M2 antibody (Sigma-Aldrich), followed by incubation with Dynabeads protein G (Invitrogen) according to the manufacturer’s instructions. Immunoprecipitated protein or input control protein was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and polyvinylidene difluoride membrane transfer. Blots were incubated with an anti-FLAG M2 antibody or a horseradish peroxidase (HRP)-conjugated anti-V5 antibody (Invitrogen). An HRP-conjugated anti-mouse secondary antibody (Cell Signaling Technology, Danvers, MA, USA) was used to detect anti-FLAG antibody signals. The signals were developed by chemiluminescence.
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