Total protein was isolated from FDB by homogenization in lysis buffer (20 mM HEPES buffer, pH 7.5, 100 mM NaCl, 1.5 mM MgCl2, 0.1% Triton X-100, 20% glycerol) containing 1 mM DTT and a protease inhibitor cocktail (Sigma) on ice. Samples were centrifuged at 20,000 g at 4 °C, and the supernatant was collected and frozen at − 80 °C until analyzed. Total protein concentration was determined using a BCA assay (Thermo Fisher Scientific). Samples were solubilized in SDS loading buffer and denatured by heating at 100 °C for 5 min. For immunoblotting, 20–30 μg total protein was loaded on bis-acrylamide gels, separated by SDS-PAGE electrophoresis, and transferred to PVDF membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% nonfat milk at room temperature for 1 h followed by overnight incubation at 4 °C in anti-SERCA1 (Thermo Fisher Scientific) or anti-calsequestrin polyclonal antibodies (Abcam ab3516). Antibodies against α-tubulin or actin (Abcam) were used as loading controls. Antibody-reactive proteins were detected with Clarity western ECL substrate (Bio-Rad, Hercules, CA, USA), imaged using an Image Lab system (Bio-Rad), and quantified by densitometry.
Ab3516
Manufactured by Abcam
Sourced in United Kingdom
Ab3516 is a laboratory equipment product offered by Abcam. It serves as a core functional tool used in various research applications. The product's detailed specifications and intended use are not available for an unbiased and factual description within the given constraints.
Automatically generated - may contain errors
Lab products found in correlation
Ab2861, by Abcam (2 mentions)
Ab135735, by Abcam (2 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Image lab system, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Ab2864, by Abcam (1 mentions)
Ab27642, by Abcam (1 mentions)
Ab16478, by Abcam (1 mentions)
Ab2865, by Abcam (1 mentions)
Ab28052, by Abcam (1 mentions)
Ab2728, by Abcam (1 mentions)
Ab9485, by Abcam (1 mentions)
Luminata forte western blotting substrate, by Merck Group (1 mentions)
Microcon centrifugal filter concentrators, by Merck Group (1 mentions)
Glutathione sepharose 4b, by GE Healthcare (1 mentions)
Phospholipid, by Avanti Polar Lipids (1 mentions)
Quantity one 4, by Bio-Rad (1 mentions)
Immobilon polyvinylidene diflouride membranes, by Merck Group (1 mentions)
10 protocols using ab3516
1
Isolation and Immunoblotting of SERCA1 and Calsequestrin
2
Protein Expression Analysis in Cardiac Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The homogenization of the samples, protein measurements, electrophoresis, primary and secondary antibody incubation, and quantification were performed following our previously established protocols (Boknik et al. 2019 (link); Gergs et al. 2021 (link); Neumann et al. 2021a (link), 2021b (link)). The following primary antibodies were used: anti-phosphorylated troponin inhibitor (P-TnI; #4004, Cell Signaling Technology Europe, Leiden, Netherlands) and anti-calsequestrin (CSQ) used as loading control (#ab3516, Abcam, Cambridge, UK).
3
Cardiac Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Crude tissue homogenate from cardiac ventricles was separated by electrophoresis and transferred onto PVDF membranes. Membranes were incubated with primary antibodies: anti‐PAD2 (1:1000, Abcam, ab16478), anti‐RyR2 (1:5000, Abcam, ab2728), anti‐phosphorylated‐S2808‐RyR (1:5000, donated by Andrew R Marks, Columbia University), anti‐dihydropyridine receptor (1:1000, Abcam, ab2864), anti‐calsequestrin 2 (1:1000, Abcam, ab3516), anti‐SR Ca2+‐ATPase 2a (SERCA 2a; 1:1000, Abcam ab2861), anti‐phospholamban (1:1000, AbCam, ab2865), α‐actin (1:1000, Abcam ab28052), anti‐citrulline antibody (1:1000, Millipore, 07–377), Malondialdehyde (MDA) antibody (1: 1000, Abcam, ab27642), GAPDH (1:1000, Abcam, ab9485), and infrared‐labeled secondary antibodies (1:5000, Licor IRDye 680 and IRDye 800). Immunoreactive bands were analyzed using the Odyssey Infrared Imaging System. Band densities were quantified with Image J and normalized to GAPDH. All the experiments were conducted blinded regarding the treatment of the mice.
4
Western Blot Analysis of Protein Signaling
5
Characterization of Calcium Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The monoclonal 34C anti-RyR1 antibody, monoclonal VIIID12 anti-CSQ1 antibody, and rabbit affinity-isolated polyclonal anti-CSQ antibody (ab 3516) were from Abcam (Cambridge, MA, USA). Polyclonal anti-junctin was a generous gift from Dr. Steven Cala (Wayne State University, MI, USA). Phospholipids were from Avanti Polar Lipids (Alabaster, AL, USA). The Pierce IP kit was from ThermoFisher Scientific (Scoresby, Vic, Australia), 45Ca2+ was from PerkinElmer (Glen Waverley, VIC, Australia), and Glutathione Sepharose 4B was from GE Healthcare (Rydalmere, NSW, Australia). The Bio-Rad DC protein determination assay and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blot apparatus and consumables were from Bio-Rad (Gladesville, NSW, Australia), and the multi-mutagenesis kit was from Stratagene (now Agilent Technologies Inc, Mulgrave, VIC, Australia). Microcon centrifugal filter concentrators were from Millipore (Bayswater, VIC, Australia). The monoclonal anti-triadin antibody (IIG12) and all other chemicals were obtained from Sigma-Aldrich (Castle Hill, NSW, Australia).
6
Right-atrial Tissue Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Right-atrial tissue samples of about 20 mg were pulverized in liquid nitrogen and ‘homogenized’ in 200 µL of ice-cold lysis buffer containing 50 mM HEPES pH 7.4, 0.1% (v/v) Tween 20, 100 mM NaCl, 2.5 mM EGTA, 10 mM glycerol-2-phosphate, 10% (v/v) glycerol, and 1 mM DTT supplemented with a cocktail of protease inhibitors (Roche). Proteins were separated by SDS–PAGE (10% acrylamide: bisacrylamide) and electrotransferred onto Immobilon polyvinylidene diflouride membranes (Millipore). Membranes were incubated with primary and secondary antibodies diluted in 5% non-fat dry milk except for DHPR blots, for which SuperBlock™ Blocking Buffer (Thermo Scientific) was used. Antibodies against SERCA (#9580, Cell Signaling Technology), calsequestrin-2 (ab3516, Abcam), DHPR (ab81980, Abcam), and NCX1 (ab135735, Abcam) were used. After a standard washing protocol, detection was performed using the appropriate horseradish peroxidase-labelled IgG and the Supersignal™ detection system (Supersignal West Dura™, Pierce). Molecular-mass standards (Bioline) were used to estimate protein size and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; MAB374, Millipore) was used as a loading control. Immunoblots were digitized (GS-800 Calibrated Densitometer; Bio-Rad) and analysed with the Quantity One 4.6.3 software (Bio-Rad).
7
Western Blot Analysis of Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were obtained by incubating cells in RIPA Lysis Buffer (Beyotin, Beijing, China) containing protease inhibitors for 30 min followed by ultrasonication and centrifugation. The supernatant containing proteins was collected. The concentration of proteins in the supernatant was determined using the BCA protein assay (Beyotime, Beijing, China). Proteins were separated in 10 or 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Merck Millipore, USA). After blocking in 5% skim milk, membranes were incubated with primary antibodies (ATP1A2, 1:1000, 16,836-1-AP, proteintech; CASQ2, 1:1000, ab3516, Abcam, UK; RYR2, 1:1000, MA3-916, Thermo Scientific, USA) overnight at 4°C and incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. The bands were detected using HRP Substrate Reagent (Thermo Fisher Scientific Inc., MA, USA) and Image Quant LAS 4000 (GE Healthcare Life Science). Protein levels of GAPDH were used as the endogenous control.
8
Immunoblotting Analysis of Cardiac Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following reagents were used: ATX-II (Alomone Labs, Jerusalem, Israel). The antibodies used were mouse anti-A2AR (sc-32261, Santa Cruz Biotechnology Inc.), rabbit anti-α-actinin (sc-17829; Santa Cruz Biotechnology Inc.), mouse anti-sarco/endoplasmic reticulum calcium-ATPase 2 (SERCA2a) (D51B11, Cell Signaling Technlogy, Danvers, MA, USA), rabbit anti-Na+/Ca2+ exchanger 1 (NCX-1) (ab135735, Abcam, Cambridge, United Kingdom), rabbit anti-calsequestrin-2 (Csq-2) (ab3516, Abcam), horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (Pierce Biotechnology, Rockford, IL, USA) and HRP-conjugated goat anti-rabbit IgG (Pierce Biotechnology, Rockford, IL, USA).
9
Cardiac Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The homogenization of the samples, protein measurements, electrophoresis, primary and secondary antibody incubation, and quantification were performed following our previously established protocols (Gergs et al. 2009 (Gergs et al. , 2019a,b;,b; Boknik et al. 2018) (link). First antibodies were rabbit polyclonal anti-calsequestrin (CSQ) antibody, #ab3516, Abcam, Cambridge, UK (diluted 1:20000), rabbit polyclonal anti-DRD1, #bs-1007R, Bioss, Woburn, MA, USA (dilution 1:500), and rabbit polyclonal anti-phospho-troponin I (P-TnI) antibody, #4004, Cell Signaling Technology Europe, Leiden, The Netherlands (diluted 1:5000).
As controls in some Western blotting experiments, cardiac homogenates from D 1 receptor knock-out mice were used. Cardiac samples of D 1 knock-out mice were kindly provided by Jean-Antoine Girault, Insern Research Director; Institute du Fer à Moulin, Paris, France.
As controls in some Western blotting experiments, cardiac homogenates from D 1 receptor knock-out mice were used. Cardiac samples of D 1 knock-out mice were kindly provided by Jean-Antoine Girault, Insern Research Director; Institute du Fer à Moulin, Paris, France.
10
Protein Expression Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We homogenized the samples, measured protein concentrations, performed electrophoresis, incubated with first primary and then secondary antibodies and quantified results as published (Gergs et al. 2009 (Gergs et al. , 2019a,b;,b; Boknik et al. 2018) . First antibodies were anti-calsequestrin (CSQ) antibody (#ab3516, abcam, Cambridge, UK, diluted 1:20,000), antiphospholamban antibody (#A010-14, Badrilla, Leeds, UK, diluted 1:5,000), and anti- sarcoplasmic calcium ATPase (SERCA2) antibody (#ab2861, abcam, Cambridge, UK, diluted 1:5,000).
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!