PKC activity in cell lysates was assayed using the PKC Kinase Activity Assay Kit (Cat: ab139437; Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions. NF-κB transcriptional activity was determined using the pGL4 luciferase reporter vector, which contains five copies of NF-κB response elements. The cells were transfected with plasmid DNA using LipofectamineTM 3000 (Invitrogen) transfection reagent. Cell lysates were collected, and luciferase activity was evaluated using the Bright-Glo™ luciferase detection assay according to the manufacturer’s protocol (Cat: E2610; Promega, Madison, WI, USA). Luciferase was measured using the Infinite 200 Microplate Reader (Tecan, Mnnedorf, Switzerland).
Pkc kinase activity assay kit
Manufactured by Abcam
Sourced in United Kingdom
The PKC Kinase Activity Assay Kit is a tool for measuring the activity of Protein Kinase C (PKC) enzymes in cell and tissue samples. It provides a quantitative assessment of PKC activity levels.
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Lab products found in correlation
Centrifuge 5415r, by Eppendorf (2 mentions)
E4030, by Promega (2 mentions)
Pka kinase activity assay kit, by Abcam (2 mentions)
Dc protein assay kit, by Bio-Rad (2 mentions)
Lipofectaminetm 3000, by Thermo Fisher Scientific (1 mentions)
Infinite 200 microplate reader, by Tecan (1 mentions)
Infinite m200, by Tecan (1 mentions)
Bca method, by Thermo Fisher Scientific (1 mentions)
Enspire, by PerkinElmer (1 mentions)
Luminoskan microplate reader, by Thermo Fisher Scientific (1 mentions)
G 6983, by Merck Group (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Halttm protease and phosphatase inhibitor single use cocktail, by Thermo Fisher Scientific (1 mentions)
Lysis buffer, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Glomax, by Promega (1 mentions)
Infinite m200 microplate reader, by Tecan (1 mentions)
Native lysis buffer, by Abcam (1 mentions)
30 protocols using pkc kinase activity assay kit
1
Evaluating PKC and NF-κB Signaling
2
PKC Kinase Activity in SKCO-15 and T84 Cells
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SKCO-15 and T84 cells were grown in 6-well plates and infected for the indicated time points. PKC Kinase Activity Assay Kit (ab139437, Abcam, Cambridge, MA) was used according to the manufacturer’s instructions.
3
Quantification of Retinal PKC Activity
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The cytoplasmic and membrane protein samples were prepared from the retinal tissues, which were isolated from the nuclei-free eyeballs of rats. PKC Kinase activity was analyzed using the PKC Kinase Activity Assay Kit (ab139437; Abcam) according to the manufacturer’s instructions. The protein concentration of the samples and the absorbance (OD at 450 nm) values for the PKC kinase activity were determined using the DR-200Bs microplate reader (Diatek, Wuxi, China). PKC activity = OD/(protein content × reaction time).
4
PKC Inhibition Assay in HepaG2 Cells
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Protein kinase C (PKC) inhibition was assayed in lysates from HepaG2 cells, using PKC Kinase Activity Assay Kit (ab139437; Abcam). Cells were grown to 90% confluency in a 60 mm dish. After removing the medium, 1 mL of lysis buffer (E4030, Promega) was applied for 10 minutes on ice. Cells were scraped, sonicated, and centrifuged at 13 000 rpm/15 minutes/4°C (Eppendorf Centrifuge 5415R; Eppendorf, Stevenage, U.K.). Then, 3 µL of cell lysate were mixed with 297 µL of Kinase Assay Buffer and aliquoted 40 µL into 0.5 mL microtubes. These aliquots were mixed with 1/100 stock solutions of carvones, resulting in final concentrations of 10 µM, 100 µM, and 1000 µM. DMSO (1% V/V) and staurosporine (1 µM) were negative and positive control, respectively. The reaction was initiated by the addition of 10 µL of reconstituted ATP, and the rest of the procedure was performed as described in the manufacturer's recommendation. Absorbance was measured at 450 nm using microplate reader Infinite M200 (TECAN, Austria). Results are expressed as % of the negative control. The cell lysate was stored at -80°C and used for performing three independent experiments.
5
Quantification of PKC Kinase Activity
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Neurospheres were disaggregated and 20,000 single cells were seeded per well. Treatments (5 μM EOF2 or vehicle) were added for 1 h prior to cell centrifugation (200 g, 5 min) and lysis. Inhibitors were added 30 min before the addition of the treatments. Protein content was measure in the lysates using the BCA method (Thermo Fisher Scientific, Rockford, IL, USA), and 1.5 μg of crude protein was used per assay. The amount of PKC kinase activity was measured in each sample using the PKC Kinase Activity Assay Kit (Abcam, Cambridge, U.K.; cat. No. ab139437), following the manufacturer’s instructions. Positive controls (20–60 ng of purified active PKC supplied by the kit) and blanks (diluent only) were included in each independent determination. Blanks were subtracted from measurements before comparisons were made.
6
Quantifying aPKC-PAR6 Complex Activity
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The activity of purified dimeric aPKC–PAR6 complex was tested using the PKC Kinase Activity Assay Kit (Abcam, Cambridge, U.K.) with a protein concentration dilution series of 5 ng, 10 ng, and 20 ng of the purified protein complex following the manufacturer’s instructions, and the standard error of the mean (SEM) was used for visualization (n = 3 replicates). As positive control, 36 ng of control protein (Abcam) was used. A multimode plate reader (EnSpire, PerkinElmer, Waltham, MA, U.S.A.) was used to measure the reaction signal.
7
Quantifying GAPDH and PKC Activity
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Before assessing GAPDH and PKC activities, a Bradford assay was performed, and the lysate volume of the samples was modified to equal the protein concentration.
For GAPDH activity, mesothelial cells were cultured in 24-well plates, and the activity of the enzyme was determined with a colorimetric GAPDH Assay (ScienCell, Carlsbad, CA, USA) according to the protocol provided by the manufacturer.
For PKC activity, mesothelial cells were cultured in six-well plates, rinsed two times with ice-cold PBS and lysed with 1 mL lysis buffer. Following lysis, the PKC activity was measured colorimetrically with a PKC Kinase Activity Assay Kit (Abcam), according to the manufacturer’s instructions.
For GAPDH activity, mesothelial cells were cultured in 24-well plates, and the activity of the enzyme was determined with a colorimetric GAPDH Assay (ScienCell, Carlsbad, CA, USA) according to the protocol provided by the manufacturer.
For PKC activity, mesothelial cells were cultured in six-well plates, rinsed two times with ice-cold PBS and lysed with 1 mL lysis buffer. Following lysis, the PKC activity was measured colorimetrically with a PKC Kinase Activity Assay Kit (Abcam), according to the manufacturer’s instructions.
8
PKA and PKC Activity Assays
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PKA and PKC activities were respectively determined using a PKA kinase activity assay kit or a PKC kinase activity assay kit (Abcam, Cambridge, UK), following the manufacturers’ protocols [12 (link)].
9
Measuring PKA and PKC Activities in Arteries
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PKA and PKC activities were, respectively, determined using a PKA kinase activity assay kit or a PKC kinase activity assay kit (Abcam, Cambridge, UK). Briefly, endothelium-denuded frozen arteries from both CT and HT animals were homogenized in a lysis buffer containing 1 mmol/L sodium vanadate, 1% SDS and pH 7.4, 0.01 mol/L Tris-HCl and centrifuged at 12,000× g for 10 min at 4 °C. The supernatant was then collected and used for the assay [35 (link),36 (link)]. Assays were performed following the manufacturers’ protocols. Protein content was measured using a DC protein assay kit (BioRad, Madrid, Spain). Results were expressed as optical density (OD) units/μg protein.
10
Quantifying PKC Activity in Cells
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Cellular PKC activities were measured using a PKC Kinase Activity Assay kit (Abcam, Cambridge, MA, UK). Cells treated with gefitinib or DMSO for 72 h were lysed in ice-cold RIPA buffer, sonicated, and centrifuged at 10,000 rpm at 4 °C for 15 min. The supernatant was assayed for PKC activity. Briefly, proteins were added to an active PKC-capturing antibody-coated 96-well plate and incubated at 30 °C for 90 min. The captured PKC proteins were treated with phospho-specific substrate antibody, washed, and then incubated with diluted anti-rabbit IgG-HRP-conjugated antibody at 25 °C for 30 min. The reacted wells were washed and incubated with TMB substrate at 25 °C for 30 min and then treated with stop solution. Absorbance was measured immediately at 450 nm using a Luminoskan microplate reader (Thermo Scientific). Data were normalized with respect to protein concentrations.
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