ccRCC tissues were resolved using a 10% polyacrylamide gel in a sodium dodecyl sulfate buffer by electrophoresis. Subsequent to being transferred onto a nitrocellulose membrane, the blots were incubated with anti-Rab25 antibody (Abcam, Cambridge, MA, USA). Binding of Rab25 antibody was revealed by chemiluminescence following incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Bio-Rad Laboratories, Hercules, CA, USA). GAPDH (Cell Signaling Technology, Inc., Danvers, MA, USA) was used as the internal control.
Horseradish peroxidase hrp conjugated goat anti mouse antibody
Manufactured by Bio-Rad
Sourced in United States
Horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody is a secondary antibody that binds to mouse primary antibodies. The HRP enzyme label can be used for detection purposes in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Cell Signaling Technology (1 mentions)
Glass bead, by Merck Group (1 mentions)
Stain free gel, by Bio-Rad (1 mentions)
Murine anti ha antibody, by Merck Group (1 mentions)
Iblot system, by Thermo Fisher Scientific (1 mentions)
Fastprep 24, by MP Biomedicals (1 mentions)
Mouse anti his antibody, by Santa Cruz Biotechnology (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Amersham ecl, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Powerwave plate reader, by Agilent Technologies (1 mentions)
Microwell maxisorp flat bottom plate, by Merck Group (1 mentions)
Clone 9b11, by Cell Signaling Technology (1 mentions)
1 step ultra tmb elisa substrate, by Thermo Fisher Scientific (1 mentions)
5 protocols using horseradish peroxidase hrp conjugated goat anti mouse antibody
1
Western Blot Analysis of Rab25 in ccRCC
2
Protein Extraction and Immunoblotting Protocol
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The bacteria were harvested 2 h after induction and washed twice with ice-cold Tris-buffered sucrose (pH 7.0, 10 mM MgCl2, 250 mM sucrose). After centrifugation, the bacteria were disrupted by glass beads (106 micron, Sigma) using a FastPrep®-24 instrument (MP Biomedicals) to obtain protein extracts. After cell disruption and centrifugation, cell-free supernatants were mixed with SDS-PAGE sample buffer. After boiling the samples for 7 min, they were applied to 10% Stain-Free gels (Biorad), followed by electrophoresis with subsequent immunoblotting using the iBlot system (Thermo Fisher Scientific) according to the instructions of the manufacturer. Protein detection was performed with the SNAP i.d. System (Millipore), using a murine anti-HA antibody (Sigma) and a horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (BioRad). Proteins were visualized using the SuperSignal West Pico chemiluminescent substrate (Pierce).
3
eVP35 IID-NP Protein Interaction Assay
4
Western Blot Analysis of HA1 Protein
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rHA1 was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the separated protein bands were visualized by Coomassie blue staining and silver staining. The proteins were transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA) and blocked with 5% skim milk in phosphate buffered saline (PBS) containing tween-20 (PBST, 1× PBS and 0.05% Tween-20) for 1 hour at room temperature (RT). The membrane was incubated with an anti-HA1 mouse monoclonal antibody (1:1,000, ATGen, Seoul, Korea) in 0.2% bovine serum albumin (BSA) in PBST after 3 washes with PBST at RT. The membranes were incubated at 4℃ overnight. Horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (1:3,000, Bio-Rad) as a secondary antibody in 5% skim milk-PBST was added after PBST washing and incubated for 1 hour at RT. After washing with PBST 3 times, the membranes were developed using a chemiluminiescent substrate (Amersham ECL, GE Healthcare, Buckinghamshire, UK) and exposing the protein side of the membrane to X-ray film.
5
Nanobody Binding Assay for US28 Mutants
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Fifty micrograms of membrane extracts of HEK293T-iUS28 or HEK293T cells transfected with the different US28 ICL mutants were coated overnight in a 96 well MicroWell MaxiSorp flat bottom plate (Sigma-Aldrich, Saint Louis, Missouri, USA). The next day, plates were washed with 1× PBS and blocked with 2% (w/v) skimmed milk in PBS for 1 h at RT. Nanobodies were diluted in 2% (w/v) skimmed milk and incubated for 1 h at RT. Nanobodies were detected with mouse-anti-Myc antibody (1:1000, Clone 9B11, Cell Signaling Technology, Leiden, The Netherlands) and horseradish peroxidase (HRP)-conjugated goat-anti-mouse antibody (1:1000, Bio-Rad). Incubations with antibodies were done for 1 h at RT. Wells were washed three times with 1× PBS between all incubation steps. Binding was determined with 1-step Ultra TMB-ELISA substrate (Thermo Fisher Scientific) and the reaction was stopped with 1 M H2SO4. Optical density was measured at 450 nm with a PowerWave plate reader (BioTek). Data were analyzed using GraphPad Prism version 8.0 (GraphPad Software, Inc., La Jolla, CA, USA).
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