Immunoblotting analysis was performed as already described elsewhere40 (link)–42 (link). Briefly, the proteins from hearts or cell lysates were homogenized in RIPA buffer (Sigma-Aldrich) containing phosphatase inhibitor (Phos-STOP, Roche) and protease inhibitor (Complete Mini EDTA-free, Roche). Equal amounts of proteins (50 mg) underwent SDS-polyacrylamide gel electrophoresis (PAGE) and were transferred to polyvinylidene difluoride membranes. Membranes were incubated with primary antibodies (anti-BNIP3L, anti-fibronectin anti-InsP3R: Santa Cruz; anti-TGF-β, anti-smad2/3, anti-Bcl-XL: CST; anti-collagen I: abcam, anti-PKCα and anti-p-PKCα: abcam) and then probed with horseradish peroxidase conjugated secondary antibodies. Blots were visualized with the use of SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Scientific, USA) and images were captured with an ImageQuant LAS 4000 (GE,USA). The densities of bands were quantified by use of Image-Quant TL software (GE Healthcare).
Anti pkcα
Manufactured by Abcam
Sourced in United States, United Kingdom, Germany
Anti-PKCα is a primary antibody that specifically recognizes the PKCα (Protein Kinase C alpha) protein. PKCα is a serine/threonine-specific protein kinase that plays a role in various cellular processes, including cell growth, differentiation, and signal transduction.
Automatically generated - may contain errors
Lab products found in correlation
Anti p pkcα, by Abcam (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Anti collagen 1, by Abcam (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Phosstop, by Roche (1 mentions)
Imagequant tl software, by GE Healthcare (1 mentions)
Complete mini edta free, by Roche (1 mentions)
Las 4000, by Cytiva (1 mentions)
Anti zo 1, by Thermo Fisher Scientific (1 mentions)
Anti ripk3, by Santa Cruz Biotechnology (1 mentions)
Anti rpe65, by Novus Biologicals (1 mentions)
Anti β amyloid clone 6e10, by BioLegend (1 mentions)
Anti p mlkl, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Anti s opsin, by Merck Group (1 mentions)
Anti m opsin, by Merck Group (1 mentions)
Anti p21, by Santa Cruz Biotechnology (1 mentions)
Anti ctbp2, by BD (1 mentions)
Anti pkcδ, by Abcam (1 mentions)
5 protocols using anti pkcα
1
Immunoblotting Analysis of Cardiac Proteins
2
Immunohistochemical Evaluation of PKCα
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To evaluate the effect of LRK on the expression of PKCα in breast tissue of HMG rats, immunohistochemistry was performed as described previously (Chen et al., 2014 (link)). Breast tissue slides were incubated with anti- PKCα (Abcam, Cambridge, MA, United States) for 12 h at 4°C. After treatment with HRP-conjugated goat anti-mouse IgG and 50 μL of streptavidin-peroxidase solutions for 30 min at RT, the sections were stained with DAB and counterstained with hematoxylin. The positive areas showed the color of brown yellow.
3
Comprehensive Immunohistochemical Analysis of Retina and Brain
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Cross-sections of the retina or brain and whole-mount retinas for immunohistochemical analysis were firstly washed several times with PBS, and then blocked in PBS containing 5% goat serum and 1% Triton x-100 for 1h at room temperature. The blocked tissues were incubated with the following primary antibodies overnight at 4°C: anti-β-amyloid, clone 6E10 (1:500; BioLegend), anti-β-amyloid, clone 4G8 (1:500; BioLegend), anti-rhodopsin, clone 1D4 (1:1000; Santa Cruz), anti-M-opsin (1:500; Millipore), anti-S-opsin (1:500; Millipore), anti-PKCα (1:200; Abcam), anti-PKCα (1:100; Invitrogen), anti-CtBP2 (1:200; BD), anti-PSD95 (1:100; CST), anti-ZO-1 (1:100; Invitrogen), anti-RPE65 (1:200; Novus), anti-p16ink4α (1:100; Proteintech), anti-p21 (1:100; Santa Cruz), anti-cleaved caspase3 (1:100; CST), anti-RIPK3 (1:100; Santa Cruz), anti-pRIPK3 (1:400; CST) and anti-pMLKL (1:100; Abcam). AlexFluro-488 or -555 conjugated secondary antibody (1:500; CST) incubation were performed for 2.5 h at room temperature. Thereafter, DAPI (1:1000, Invitrogen) were used to label the nuclear for 10 min at room temperature.
4
Westernblot Analysis of Cardiac Proteins
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Both H9C2 cells and myocardial tissues were harvested for Western blot analysis (Western Blot Detection Kit, Elabscience) following standard protocol according to the manufacturer’s instructions and Towbin system buffer was used. Primary antibodies used in Western blot were as follows: anti-GLP-1R (1:200, Abcam, UK), anti-Akt (1:200, Abcam, UK), anti-p-Akt (1:200, Abcam, UK), anti-PI3K (1:500, Santa Cruz, USA), anti-PKC (1:500, Abcam, UK), anti-PKCα (1:500, Abcam, UK), anti-PKCβ (1:500, Abcam, UK), anti-PKCγ (1:500, Abcam, UK), anti-PKCδ (1:500, Abcam, UK) and anti-β-actin (1:500, Santa Cruz, USA). Secondary antibodies were horseradish peroxidase-conjugated goat anti-rabbit lgG (Santa Cruz, USA) at 1:5000 dilution. The results were visualized using an enhanced chemiluminescence system (ECL, Amersham). Densitometric analysis was conducted using ImageJ software (NIH, USA).
5
Immunohistochemistry Protocol for Retinal Analysis
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The protocol for immunohistochemistry was previously described.25 (link) The retinas were incubated in PBS with 1% Triton X-100 and 0.5% Tween 20 for 1 h at room temperature and in 4% BSA for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies: anti-FLAG (1:500, Merck, Darmstadt, Germany) and anti-PKCα (1:100, Abcam, Cambridge, UK) in blocking buffer. Secondary anti-rabbit, conjugated with Alexa TM488 or 594 (1:1000; Abcam), were applied for 1 h at room temperature.
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