Changes in the expression of ion channels and Ca2+-modulated proteins were investigated through Western blotting. RVOT tissues were homogenized and centrifuged in buffer systems. Equal amounts of total protein were separated through 5% or 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The resultant protein bands were electrophoretically transferred onto polyvinylidene difluoride membranes. For the immunofluorescence-based detection of gap junction proteins, all blots were stained with primary antibodies against sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a), Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylated at Thr286 (pCaMKII), RyR2, total phospholamban (Thermo Fisher Scientific, Rockford, IL, USA), phosphorylated RyR at Ser2808, phospholamban phosphorylated at Thr17 (Badrilla, UK), catalytic subunit of protein kinase A (BD BioSciences, USA), Cav1.2, NLRP3, nuclear factor (NF)-κB, IL-1β, and glyceraldehyde-3-phosphate dehydrogenase (MBL, Japan); all secondary antibodies were conjugated with horseradish peroxidase. Bound antibodies were detected using an enhanced chemiluminescence detection system and were analyzed using the AlphaEaseFC software. To ensure equal protein loading, all target bands were normalized to a band of glyceraldehyde-3-phosphate dehydrogenase.
P camkii
Manufactured by Thermo Fisher Scientific
Sourced in United States
P-CaMKII is a laboratory instrument designed for the detection and quantification of phosphorylated calcium/calmodulin-dependent protein kinase II (P-CaMKII). P-CaMKII is an important signaling protein involved in various cellular processes. The core function of this product is to provide reliable and accurate measurements of P-CaMKII levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Serca2a, by Thermo Fisher Scientific (1 mentions)
Bm0627, by Boster Bio (1 mentions)
Ab9080, by Abcam (1 mentions)
P ryr2, by Abcam (1 mentions)
Passive lysis buffer (plb), by Cell Signaling Technology (1 mentions)
P mlkl, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Anti ripk3, by Novus Biologicals (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Camkii, by Abcam (1 mentions)
Ripk1, by Cell Signaling Technology (1 mentions)
Gapdh, by Merck Group (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
3 protocols using p camkii
1
Western Blot Analysis of Ion Channels and Ca2+ Proteins
2
Western Blot Analysis of Cardiac Proteins
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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).
3
Western Blot Analysis of Cardiomyocyte Proteins
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Lysis buffer was used to harvest the protein samples from cardiomyocytes (Beyotime, Shanghai, China) and phenylmethanesulfonyl fluoride (PMSF, 100:1 in volume). The proteins (about 50 μg) were then separated by SDS-PAGE, followed by transferring to a polyvinylidene fluoride (PVDF) membrane (Millipore, Kenilworth, NJ, USA). Next, being blocked for 2 h with skim milk (5%) at room temperature, membranes’ incubation was made overnight at 4 °C with relevant primary anti-RIPK3 (1:1000, Novusbio, Littleton, CO, USA); RIPK1, MLKL, p-MLKL, caspase 3 and cleaved caspase 3 (1:1000, Cell Signaling Technology, Danvers, MA, USA); phospholamban (PLB, 1:1000), phosphorylation-PLB Thr 17 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA); phosphorylation-PLB Ser 16 (1:800, Merck KGaA, Darmstadt, Germany); ox-CaMKII (CaMKII oxidation, 1:1000, Millipore, Kenilworth, NJ, USA); p-CaMKII (1:2000, Thermo Fisher Scientific, Rockford, IL, USA); CaMKII (1:1000, Abcam, Cambridge, UK); GAPDH (1:5000, Sigma-Aldrich, St. Louis, MO, USA) and β-tubulin (1:3000, CMCTAG, Milwaukee, WI, USA) antibodies. Afterward, HRP-conjugated IgG was employed to incubate the membranes (1:5000, ZSbio, Beijing, China) for another 2 h. Protein bands were visualized by the enhanced chemiluminescence (ECL, Thermo Fisher Scientific, Rockford, IL, USA).
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