The following antibodies were used: anti-IL-32 antibody (provided by Prof. Do-Young Yoon), anti-β-actin antibody, anti-HA antibody, anti-GFP antibody, anti-PKCδ antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) anti-Flag antibody (Sigma Aldrich, St. Louis, MO, USA), anti-Myc-Tag antibody, and anti-PARP antibody (Cell Signaling Technology, Danvers, MA, USA).
Anti pkcδ antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Anti-PKCδ antibody is a laboratory research tool used to detect and study the Protein Kinase C delta (PKCδ) protein in various biological samples. PKCδ is a serine/threonine-protein kinase involved in numerous cellular processes. The antibody can be used in techniques such as Western blotting, immunoprecipitation, and immunohistochemistry to identify and quantify the PKCδ protein.
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Lab products found in correlation
Anti gfp antibody, by Santa Cruz Biotechnology (1 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Anti myc tag antibody, by Cell Signaling Technology (1 mentions)
Anti parp antibody, by Cell Signaling Technology (1 mentions)
Anti ha antibody, by Santa Cruz Biotechnology (1 mentions)
Catalase, by Merck Group (1 mentions)
Bay 41 2272, by Enzo Life Sciences (1 mentions)
Bodipy c12 sphingomyelin, by Merck Group (1 mentions)
Epigallocatechin gallate (egcg), by Merck Group (1 mentions)
Desipramine, by Merck Group (1 mentions)
Superoxide dismutase sod, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Biowest (1 mentions)
Penicillin, by Meiji Seika Pharma (1 mentions)
Streptomycin, by Meiji Seika Pharma (1 mentions)
Rpmi 1640, by Nissui Pharmaceutical (1 mentions)
Normal rabbit igg antibody, by Santa Cruz Biotechnology (1 mentions)
Multimacs protein g kit, by Miltenyi Biotec (1 mentions)
μmacs protein g microbeads, by Miltenyi Biotec (1 mentions)
Chelerythrine, by Merck Group (1 mentions)
7 protocols using anti pkcδ antibody
1
Antibody Characterization Protocol
2
Evaluating PKCδ Phosphorylation Modulators
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Penicillin and streptomycin were purchased from Meiji Seika Pharma (Tokyo, Japan), fetal calf serum (FCS) was obtained from Biowest (Nuaillé, France). RPMI-1640 was obtained from Nissui Pharmaceutical Co. Ltd. (Tokyo, Japan). Catalase, EGCG, NS2028, superoxide dismutase (SOD), BODIPY-C12-sphingomyelin (SM) and desipramine were obtained from Sigma-Aldrich. Anti-PKCδ antibody was provided by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-PKCδ antibody at Ser664 antibodies was purchased from Abcam. Vardenafil was provided by TRC (Toronto, Canada). Bay 41-2272 was obtained from Enzo Life Sciences (Villeurbanne, France).
3
Immunoprecipitation of GST- and GFP-tagged Proteins
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For immunoprecipitation procedures, 2 x 106 cells were seeded in 10 cm Petri dishes. Transfections were performed as described previously [36 (link)]. Transfection controls were incubated with growth medium without serum or penicillin-streptomycin. For TPA-stimulation, 16 nM TPA was added to the cells after transfection and 16 h after transfection, cells were collected and lysed. Immunoprecipitations were performed using MACS Separation Columns together with μMACS GST Isolation Kit for GST-tagged proteins and μMACS GFP-Tagged Protein Isolation Kit for GFP-tagged proteins (Miltenyi Biotec). For PKCδ-immunoprecipitations, 1 μg anti-PKCδ antibody (Santa Cruz) was used together with MultiMACS protein G kit and μMACS Protein G Microbeads (Miltenyi Biotec) with 1 μg normal rabbit IgG-antibody (Santa Cruz) as control. All immunoprecipitations were performed as described in manufacturer’s protocol with the exception of GST-immunoprecipitations where binding reactions were incubated with beads for 90 min. For all Western blots performed with samples from immunoprecipitation, 2 % of each sample was loaded for input fractions and 48 % was loaded for IP fractions.
4
Modulation of JNK Signaling in Tissues
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All drugs were of the highest purity available commercially. L-NAME, indomethacin, chelerythrine, Go6976, safingol, ruboxistaurin, and SP600125 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Rottlerin and ML-7 hydrochloride were purchased from Calbiochem (San Diego, CA, USA). DMT was donated by Orion Pharma (Turku, Finland). Anti-phospho-stress activated protein kinase/JNK (Thr183/Tyr185) and anti-JNK antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-PKC-δ antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). indomethacin, Go6976, safingol, rottlerin, ruboxistaurin, and SP600125 were dissolved in dimethyl sulfoxide (final organ bath concentration: 0.1%). All other drugs were dissolved and diluted in distilled water unless otherwise stated.
5
Generation of PKCδ-silenced HCT116 Cell Lines
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HCT116 cells were seeded in 24-well plates at 6 × 104 cells/well for 24 h. For the generation of stable cell lines, cells were co-transfected with 1 μg of control pSuper plasmid (control; mock) or 900 ng of pSuperPKCδ.RNAi [Addgene plasmid #10819;48 (link)] and 100 ng of pSUPERpuro plasmids, and 2 μg/ml Lipofectamine 2000 (Invitrogen) for 72 h. Stable transfectants were selected with 3 μg/ml puromycin (Sigma-Aldrich) for 4 weeks. Clones were screened for PKCδ expression silencing by Western blot using an anti-PKCδ antibody (Santa Cruz Biotechnology, Frilabo, Porto, Portugal) (Supplementary Fig. S3 ).
6
Immunoprecipitation and Western Blot Analysis of Phospho-PKCδ
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After neutrophils were incubated with DP or medium at 37°C, the cells was lysed in lysis buffer (20 mM Tris HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerol phosphate, and 1 tablet of protease inhibitor). Following incubation on ice for 20 minutes, the detergent-insoluble materials were sedimented by centrifugation at 12,000 g for 15 min at 4°C. The supernatants were then pre-cleared by incubation with 10 µl of Protein A/G Plus agarose (Santa Cruz Biotechnology). For immunoprecipitation, samples were incubated with 1 µg/ml of anti-PKCδ antibodies (Santa Cruz Biotechnology) for 2 hrs at 4°C, followed by incubation with 20 µl of Protein A/G Plus agarose for 1 hr at 4°C. The immunoprecipitates were then washed three times with ice-cold lysis buffer and boiled with 20 µl of Laemmli buffer for 4 minutes, after which Western blotting was performed using anti-phospho-PKCδ or anti-PKCδ antibodies.
7
Dissecting EGF Signaling Regulation
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