Protein concentration was determined using the Pierce BCA protein assay (Thermo-Fisher). Before undergoing SDS-PAGE, samples were boiled in Laemmli sample buffer for 5 min at 95°C. Proteins were separated in a resolving phase polymerized from 7%–9% acrylamide, then transferred to a polyvinylidene difluoride membrane at 24 V for 1–2 h at 4C. Membranes were blocked in 5% milk or BSA and incubated with anti-CaMKIIa (1:4000, CBa2, available at Invitrogen but made in house), pT286-CaMKII (1:2500, Phospho-Solutions), anti-GluN2B (1:1000, Cell Signaling), DAPK1 (1:800, Sigma-Aldrich), followed by either Amersham ECL goat anti-mouse HRP-linked secondary 1:10000 (GE Healthcare) or goat anti-rabbit horseradish peroxidase conjugate 1:10000 (Bio-Rad). Blots were developed using chemiluminescence (Super Signal West Femto, Thermo-Fisher), imaged using the Chemi-Imager 4400 system (Alpha-Innotech), and analyzed by densitometry (ImageJ). Variations in sample loading were corrected using b-actin (1:2000, Cell Signaling) as a loading control. Relative band intensity was normalized as a percent of control conditions on the same blot, which was set at a value of one to allow for comparison between multiple experiments.
Goat anti rabbit horseradish peroxidase conjugate
Manufactured by Bio-Rad
Sourced in Japan
The Goat anti-rabbit horseradish peroxidase conjugate is a secondary antibody conjugate used in immunoassays. It is composed of goat-derived polyclonal antibodies directed against rabbit immunoglobulins, which are covalently linked to the enzyme horseradish peroxidase. This conjugate can be used to detect and quantify the presence of rabbit primary antibodies in various immunological techniques.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west femto, by Thermo Fisher Scientific (3 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (3 mentions)
Pt286 camkii, by PhosphoSolutions (3 mentions)
Amersham ecl goat anti mouse hrp linked secondary, by GE Healthcare (3 mentions)
Chemi imager 4400 system, by Bio-Techne (3 mentions)
Amersham ecl, by Cytiva (3 mentions)
Anti glun2b, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Anti camkiia, by PhosphoSolutions (2 mentions)
Dapk1, by Merck Group (2 mentions)
Mini protean tgx precast gel, by Bio-Rad (2 mentions)
Mouse 3t3 l1 preadipocytes, by American Type Culture Collection (2 mentions)
Trans blot turbo transfer pack, by Bio-Rad (2 mentions)
Tris glycine sds buffer, by Bio-Rad (2 mentions)
Ripa buffer, by Nacalai Tesque (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Anti gst, by Merck Group (1 mentions)
Anti camkiiα, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
7 protocols using goat anti rabbit horseradish peroxidase conjugate
1
Western Blot Analysis of CaMKII
2
Western Blot Analysis of CaMKII
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein concentration was determined using the Pierce BCA protein assay (Thermo-Fisher). Before undergoing SDS-PAGE, samples were boiled in Laemmli sample buffer for 5 min at 95°C. Proteins were separated in a resolving phase polymerized from 7%–9% acrylamide, then transferred to a polyvinylidene difluoride membrane at 24 V for 1–2 h at 4C. Membranes were blocked in 5% milk or BSA and incubated with anti-CaMKIIa (1:4000, CBa2, available at Invitrogen but made in house), pT286-CaMKII (1:2500, Phospho-Solutions), anti-GluN2B (1:1000, Cell Signaling), DAPK1 (1:800, Sigma-Aldrich), followed by either Amersham ECL goat anti-mouse HRP-linked secondary 1:10000 (GE Healthcare) or goat anti-rabbit horseradish peroxidase conjugate 1:10000 (Bio-Rad). Blots were developed using chemiluminescence (Super Signal West Femto, Thermo-Fisher), imaged using the Chemi-Imager 4400 system (Alpha-Innotech), and analyzed by densitometry (ImageJ). Variations in sample loading were corrected using b-actin (1:2000, Cell Signaling) as a loading control. Relative band intensity was normalized as a percent of control conditions on the same blot, which was set at a value of one to allow for comparison between multiple experiments.
3
Western Blot Analysis of CaMKII
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western analysis was performed similar as described previously (Cook et al., 2021 (link); Tullis et al., 2020 (link)). Protein concentration was determined using the Pierce BCA protein assay (Thermo-Fisher). Before undergoing SDS-PAGE, samples were boiled in Laemmli sample buffer for 5 min at 95°C. Proteins were separated in a resolving phase polymerized from 7.5% acrylamide, then transferred to a polyvinylidene difluoride membrane at 24 V for 1–2 h at 4°C. Membranes were blocked in 5% milk or BSA and incubated with anti-CaMKIIα (1:4000, CBα2, available at Invitrogen but made in house), pT286-CaMKII (1:2500, Phospho-Solutions), anti-GST (1:1000, Millipore), followed by either Amersham ECL goat anti-mouse HRP-linked secondary 1:10000 (GE Healthcare) or goat anti-rabbit horseradish peroxidase conjugate 1:10000 (Bio-Rad). Blots were developed using chemiluminescence (Super Signal West Femto, Thermo-Fisher), imaged using the Chemi-Imager 4400 system (Alpha-Innotech), and analyzed by densitometry (ImageJ). Phospho-signal was corrected to total protein. Relative band intensity was normalized as a percent of control conditions on the same blot, which was set at a value of one to allow for comparison between multiple experiments.
4
Osteoclast Differentiation and Activation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
α‐Minimal essential medium (α‐MEM), penicillin, streptomycin, foetal bovine serum (FBS), qPCR SuperMix UDG kit were purchased from Invitrogen (Carlsbad, CA). Lipopolysaccharides (LPS), macrophage monocyte colony‐stimulating factor (M‐CSF), methylthiazolyldiphenyl‐tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), TRAP solution, Acid Phosphatase Liquicolor Assay kit, Hoechst 33342, ELISA kits, alamar blue reagent and phalloidin conjugate solution were purchased from Sigma‐Aldrich. Anti‐NFATc1 (H‐110), anti‐Ap1 (Sc‐57761) and anti–c‐Fos (H‐125) monoclonal antibodies were purchased from Santa Cruz Biotechnology. Anti–TNF‐α, anti‐RANKL, anti‐OPG, anti–IL‐1β, anti–IL‐6 and anti–TGF‐β were purchased from Cell Signaling Technology. NucleoSpin for RNA extraction kit was purchased from Macherey–Nagel. Toluidine blue stain was purchased from BioGenex. FastStart Essential DNA Green Master was purchased from Roche. Goat anti‐rabbit horseradish peroxidase conjugate was purchased from Bio‐Rad.
5
Mouse Preadipocyte and Macrophage Cell Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse 3T3-L1 preadipocytes were obtained from the American Type Culture Collection CL-173 (Manassas, VA, USA) , and RAW264.7 macrophages from DS Pharma Biomedical Co., Ltd. (Osaka, Japan) . Sepasol ® -RNA II Super and RIPA buffer were purchased from Nacalai Tesque Inc. (Kyoto, Japan) . The High Capacity RNA-to-cDNA Kit and Taqman ® Gene Expression Assays were purchased from Thermo Fisher Scientific K.K. (Applied Biosystems, Tokyo, Japan) . The rabbit anti-IL-6 antibody (D5W4V, #12912) and anti-α-Tubulin antibody (#2144) were purchased from Cell Signaling (Tokyo, Japan) . In addition, 10％ Mini-PROTEAN TGX Precast Gels, Tris/Glycine/SDS Buffer, Trans-Blot ® Turbo TM Transfer Pack, 10×Tris-buffered saline, 0.05％ Tween 20 solution Clarity TM Western ECL substrate and goat anti-rabbit horseradish peroxidase conjugate were purchased from BIO-RAD (Tokyo, Japan) .
6
Nontraditional SDS-PAGE for Ceruloplasmin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nontraditional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE, without boiling samples) was performed to separate apo and holoCp based on previous work by our own and other laboratories (Sato and Gitlin 1991 (link); Middleton and Linder 1993 (link); Hirano et al. 2005 (link)). Nontraditional and traditional SDS‐PAGE (with boiling) was carried out in 7.5% acrylamide resolving gels and transferred to PVDF membranes, followed by immunoblotting with primary rabbit anti‐mouse Cp antibody (Biomatik; Wilmington, DE) and goat anti‐rabbit‐horse radish peroxidase conjugate (Bio‐Rad, Hercules, CA), developing with peroxide and Luminal Enhancer solution from Thermo Scientific (Rockford, IL). Images were captured by Kodak Image Station 4000R (Molecular Bioimaging, Upland, CA).
7
Mouse Preadipocyte and Macrophage Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse 3T3-L1 preadipocytes were obtained from the American Type Culture Collection CL-173 (Manassas, VA, USA) , and RAW264.7 macrophages from DS Pharma Biomedical Co., Ltd. (Osaka, Japan) . RIPA buffer was purchased from Nacalai Tesque Inc. (Kyoto, Japan) . The rabbit anti-NF-κB antibody and anti-GAPDH antibody were purchased from Cell Signaling (Tokyo, Japan) . In addition, 10％ Mini-PROTEAN TGX Precast Gels, Tris/Glycine/SDS Buffer, Trans-Blot ® Turbo TM Transfer Pack, 10×Tris-buffered saline, 0.05％ Tween 20 solution Clarity TM Western ECL substrate and goat anti-rabbit horseradish peroxidase conjugate were purchased from BIO-RAD (Tokyo, Japan) .
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!