The common procedure for western blot analysis was described in a previous publication.18 (link) The density cof the immunoreactive bands was analyzed using ImageJ software (NIH, Bethesda, MD). In the western analysis, β-actin was used as a loading control. Primary antibodies used in this study are as follows: CaMKII (santa Cruze Biotech, SC-9035), p-CaMKII (T287, Thermo Fisher Scientific, PA5-37833), RyR2 (Millipore, AB9080), p-RyR2 (S2808, Abcam, ab59225), p-RyR2 (S2814, badrilla, A010-31), PLB (Cell signaling Technology, 8495), p-PLB (Thr17, Badrilla, A010-13), SERCA2 (Abcam, ab150435), Kv4.2 (Sigma, SAB5200070), Kv4.3 (Sigma, SAB5200076), Cav1.2 (Thermo Fisher Scientific, PA5-23015), CX43 (Abcam, ab11370), and β-actin (Boster, BM0627).
Passive lysis buffer (plb)
Manufactured by Cell Signaling Technology
Sourced in France
The PLB is a specialized lab equipment designed for cell signaling analysis. It serves as a platform for the detection and quantification of various cellular signaling molecules, including proteins, phosphoproteins, and other biomolecules. The PLB provides a reliable and accurate method for researchers to study the complex signaling pathways within cells.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Bm0627, by Boster Bio (1 mentions)
Ab9080, by Abcam (1 mentions)
P ryr2, by Abcam (1 mentions)
P camkii, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti chicken igg, by Thermo Fisher Scientific (1 mentions)
Serca2a, by Abcam (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Ecl advance western blotting detection kit, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Hrp labeled secondary antibody, by Jackson ImmunoResearch (1 mentions)
Gradient gel, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
4 protocols using passive lysis buffer (plb)
1
Western Blot Analysis of Cardiac Proteins
2
Cardiac Protein Expression Analysis
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Primary antibodies used were: SLN for Western blot (Merck Millipore, #ABT13), SERCA2a (Abcam, #ab2861), PLB (Cell Signaling, #14562), phospho-PLB(Ser16) (Santa Cruz, SC-12963-R), phospho-PLB(Thr17) (Santa Cruz, SC-17024-R), FLAG (Sigma, F3165), GAPDH (Santa Cruz, sc-47724). Secondary antibodies for immunoblotting were HRP-conjugated donkey anti-chicken (Invitrogen), rabbit anti-mouse or goat anti-rabbit IgG (Jackson ImmunoResearch). Secondary antibodies for immunofluorescence were Alexa Fluor 488 conjugated goat anti-rabbit IgG, Alexa Fluor 568-conjugated goat anti-mouse IgG, Alexa Fluor 488-conjugated goat anti-chicken IgG (Life Technologies).
3
Western Blot Analysis of Mitochondrial Proteins
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Total mitochondrial proteins were resolved on 4–15% Tris-glycine SDS-PAGE gels and electroblotted onto polyvinylidene fluoride membranes (Bio-Rad, Marnes La Coquette, France). Following electrotransfer, membranes were blocked for 1 h at room temperature in 5% BSA-PBST (10 mM Tris-HCl, pH 8.0/150 mM NaCl/0.1% Tween-20). Next, membranes were incubated overnight at 4 °C with primary antibody. The day after, the membranes were washed six times with PBST and incubated with peroxidase-conjugated secondary antibody at room temperature for 1 h. Peroxidase activity was detected with enhanced chemiluminescence (ECL Advance Western Blotting Detection Kit; Thermo Scientific, Villebon sur Yvette, France). For protein detection, the following antibodies were used: sAC (Abcam Cambridge, UK; CEP Biotech, Tamarac, FL, USA), Epac1, 2 (Cell Signaling, Danvers, MA, USA), ANT (Abcam), GAPDH (Cell Signaling), VDAC (Genosphere, Paris, France), TnI (Cell Signaling), PLB (Cell Signaling) and MCU (Biorbyt, Berkeley, CA, USA).
4
Western Blotting of Muscle Proteins
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Western blotting was performed according to standard procedures as previously described (Whitehead et al. 2015 ). Briefly, muscles were homogenized in a PBS buffer containing EDTA (5 mmol/L), protease (Thermo Scientific) and phosphatase (Roche) inhibitor cocktails, and 1% Triton X‐100. Samples were loaded onto 4–20% gradient gels (Bio‐Rad) and transferred to PVDF membranes (Millipore). Membranes were blocked for 1 to 2 h with 5% skim milk in PBST or 2.5% BSA in PBST for phosphorylated proteins, and then incubated with primary antibodies in blocking buffer for 1 h at room temperature or overnight at 4°C. Primary antibodies used were; p‐PLB‐S16 (Millipore Cat# 07‐052, RRID:AB_310352), PLB (Cell Signaling Technology Cat# 8495S, RRID:AB_10949105), SERCA2 ATPase (Sigma‐Aldrich Cat# S1314, RRID:AB_261436) and GAPDH (Santa Cruz Biotechnology Cat# sc‐25778, RRID:AB_10167668). HRP‐labeled secondary antibodies (Jackson ImmunoResearch) were incubated for 1 h at room temperature. Bands were detected using a FluorChem M imaging system (Protein Simple).
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