The protocol was according to that described by Wu at al. (84 (link)). Briefly, crude protein lysates (100 μg) containing YFP-tagged “prey” proteins were loaded onto an SDS (bis-Tris) polyacrylamide (4% to 12%) gel and transferred to a polyvinylidene difluoride (PVDF) membrane followed by denaturing and renaturing steps. The membrane was blocked with 5% milk and incubated with 5 μg (1 μg/ml) purified recombinant GST-Rsr1 “bait” protein in PBS with 5 mM MgCl2 for 3 h at room temperature (RT). GST-Rsr1 was detected with horseradish peroxidase (HRP)-conjugated anti-GST antibody (8-326; Thermo Fisher Scientific) using a chemiluminescence two-component kit (Pierce ECL Western blotting substrate; Thermo Fischer Scientific). The membrane was stripped using stripping buffer (Thermo Fisher Scientific) for 15 min at RT, and YFP-labeled proteins were detected by anti-GFP antibody (7.1 and 13.1; Roche), followed by HRP-conjugated anti-mouse antibody (7076; Cell Signaling Technology).
Hrp conjugated anti mouse antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The HRP-conjugated anti-mouse antibody is a secondary antibody that is coupled with horseradish peroxidase (HRP). It is designed to detect and visualize primary antibodies that have been raised in mouse hosts. This antibody can be used in various immunodetection techniques, such as Western blotting, ELISA, and immunohistochemistry, to amplify the signal from the target antigen.
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Lab products found in correlation
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Stripping buffer, by Thermo Fisher Scientific (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Anti gfp antibody, by Roche (1 mentions)
C digit blot scanner, by LI COR (1 mentions)
Bca protein assay, by Merck Group (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Sc 32268, by Santa Cruz Biotechnology (1 mentions)
Chemidoc mp image system, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
4 protocols using hrp conjugated anti mouse antibody
1
Yeast Protein Interaction Assay
2
Targeted delivery of imatinib and silver nanoparticles for KU812 leukemia cell treatment
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KU812 leukemia cells (106 cells/mL) were incubated with 500 ng/mL of empty PCL NPs, 150 nM of IM, 250 nM of AgNPs, 100 nM of IM/AgNPs free combination in the medium, 100 nM of IM-PCL NPs, and 50 nM of HC-NPs. After 7 days of incubations with the different formulations, the samples were washed in PBS 1× at 4 °C and resuspended in lysis buffer containing a protease inhibitor cocktail on ice for 30 min. Protein concentration was determined using the BCA protein assay (Sigma-Aldrich). Protein bands were separated on 10% (w/v) SDS-polyacrylamide gels, and immunoblotting was performed using nitrocellulose membrane (Amersham Hybond ECL Nitrocellulose Membrane-GE, Abcam). Primary incubations with specific primary antibody directed against anti-c-ABL 1:1000 (clone K-12, Santa Cruz Biotechnology Inc., CA, USA) and anti-βactin 1:5000 (Sigma-Aldrich) were performed overnight. Secondary incubations were for 1 h with HRP-conjugated anti-mouse antibody (Cell Signaling). Proteins were visualized by chemiluminescence (Clarity—Western ECL Substrate, BioRad) using the C-DiGit blot scanner (LI-COR, Cornaredo Milano, Italy). Densitometric analysis was performed using Image J software on the western blots, normalizing to β-actin used as control.
3
Tau Acetylation Verification by Western Blot
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Tau acetylation was verified by western blot against acetyl-lysine. 100-ng samples of Ac-Tau, wt Tau and BSA were loaded on a 4–20% gradient SDS-PAGE gel (Mini-PROTEAN TGX Precast Gels, Bio-Rad). After electrophoresis, the gel was transferred to a polyvinylidene fluoride (PVDF) membrane using the Trans-Blot Turbo Transfer System, following the manufacturer’s protocols (Bio-Rad). After incubation with 5% (w/v) nonfat milk in TBS-T (150 mM NaCl, 0.1% Tween-20, 20 mM Tris-HCl, pH 7.5) at RT for 2 h, the membrane was incubated with antibody against acetyl-lysine (1:100 in 1% nonfat milk/TBS-T; sc-32268, Santa Cruz Biotech, Dallas, TX, USA) overnight at 4 °C. The membrane was washed six times for 10 min with TBS-T and incubated with HRP-conjugated anti-mouse antibody (1:1000; #7076, Cell Signaling, Danvers, MA, USA) at RT for 30 min. The membrane was washed six times and developed with Clarity Western ECL Substrate according to the manufacturer’s protocols (Bio-Rad). Chemiluminescent signals were measured using ChemiDoc MP Image System (Bio-Rad).
4
Immunogold Labeling of Tau and Glutamate Receptors
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Immunohistochemical reactions at the electron microscopic level were conducted using the pre-embedding immunogold method, following the procedure described earlier by Martín-Belmonte et al. (2020a).
The primary antibodies used were: a mouse monoclonal anti-p-Tau PHF1 (Ser396/Ser404); a mouse monoclonal anti-total Tau (clone T46, Sigma-Aldrich, St. Louis, MO, USA, Cat# T9450, RRID: AB_477595); a rabbit pan-GluA1 subunit antibody (aa. 841–907 of mouse AMPAR, Frontier Institute Co., Sapporo, Japan, Cat# Rb-Af690); a polyclonal rabbit antibody anti-GluA2/3 (Chemicon, Temecula, CA, USA, Cat# AB1506), a rabbit anti-mGlu5 polyclonal antibody (aa. 1144–1171 of mouse mGlu5; Frontier Institute Co., Cat# Rb-Af300, RRID: AB_2571802). The preparation, purification, and full characterization of the rabbit anti-GluA1 polyclonal antibody have been described previously (Fukaya et al., 2006). The specificity of the rabbit anti-mGlu5 polyclonal antibody has been validated in mGlu5 KO mice (García-Negredo et al., 2014). The secondary antibodies used were as follows: HRP-conjugated anti-mouse antibody (Cell Signaling Technology, Cat# 7076, RRID: AB_330924), goat anti-mouse and anti-rabbit IgG coupled to 1.4 nm gold antibodies (1:100; Nanoprobes Inc., Stony Brook, NY, USA).
The primary antibodies used were: a mouse monoclonal anti-p-Tau PHF1 (Ser396/Ser404); a mouse monoclonal anti-total Tau (clone T46, Sigma-Aldrich, St. Louis, MO, USA, Cat# T9450, RRID: AB_477595); a rabbit pan-GluA1 subunit antibody (aa. 841–907 of mouse AMPAR, Frontier Institute Co., Sapporo, Japan, Cat# Rb-Af690); a polyclonal rabbit antibody anti-GluA2/3 (Chemicon, Temecula, CA, USA, Cat# AB1506), a rabbit anti-mGlu5 polyclonal antibody (aa. 1144–1171 of mouse mGlu5; Frontier Institute Co., Cat# Rb-Af300, RRID: AB_2571802). The preparation, purification, and full characterization of the rabbit anti-GluA1 polyclonal antibody have been described previously (Fukaya et al., 2006). The specificity of the rabbit anti-mGlu5 polyclonal antibody has been validated in mGlu5 KO mice (García-Negredo et al., 2014). The secondary antibodies used were as follows: HRP-conjugated anti-mouse antibody (Cell Signaling Technology, Cat# 7076, RRID: AB_330924), goat anti-mouse and anti-rabbit IgG coupled to 1.4 nm gold antibodies (1:100; Nanoprobes Inc., Stony Brook, NY, USA).
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