200 µg of protein from TG of 3-month old TNF-α cTg and control mice was dissolved in T-PER buffer and immunoprecipitated using 4 µg of anti-Cdk5 (C8) antibody (Santa Cruz Biotechnology, Dallas, TX). Briefly, immunoprecipitated proteins (IP) were washed three times in cold PBS, and twice in kinase buffer (20 mM Tris HCl (pH 7.4), 10 mM MgCl2, 1 mM EDTA). Then, the IP was mixed with the kinase assay mixture (100 mM Tris HCl (pH 7.4), 50 mM MgCl2, 5 mM EDTA, and 5 mM DTT) plus 5 units of (γP32)-ATP, and 5 µg of histone H1 as a substrate. The kinase assays were carried out for 45 minutes at 30°C, and the kinase activity reaction was stopped by adding loading buffer and boiling for 10 min at 95°C. The kinase reaction was electrophoresed on a 12% polyacrylamide gel, and then the gels were exposed to X-ray films for 1 to 3 hours at −80°C. The incorporation of P32 to histone H1 was quantified by measuring band intensity using an image analysis system with ImageJ 1.46r software.
Anti cdk5 antibody c8
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Anti-CDK5 antibody (C8) is a laboratory research tool used to detect and study the CDK5 protein in various biological samples. It is a highly specific antibody that recognizes the CDK5 protein, a member of the cyclin-dependent kinase family, which plays a crucial role in various cellular processes.
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Lab products found in correlation
Scion image alpha 4, by Techcomp Instruments (1 mentions)
Whatman, by Cytiva (1 mentions)
Alexa fluor 488 conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Anti actin antibody, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (1 mentions)
Phos tag acrylamide, by Fujifilm (1 mentions)
Protein a g sepharose, by GE Healthcare (1 mentions)
Hrp conjugated swine anti rabbit igg, by Agilent Technologies (1 mentions)
Anti myc antibody 4a6, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated goat antimouse igg, by Agilent Technologies (1 mentions)
Anti app, by Merck Group (1 mentions)
Anti p35 c 19, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh, by Novus Biologicals (1 mentions)
Ecl plus detection kit, by Cytiva (1 mentions)
Trans blot apparatus, by Bio-Rad (1 mentions)
Anti bace1 d10e5, by Cell Signaling Technology (1 mentions)
Hybond, by Cytiva (1 mentions)
6 protocols using anti cdk5 antibody c8
1
Cdk5 Kinase Activity Assay
2
Cdk5 Kinase Activity Assay
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As reported earlier, 300 μg of protein from brain cortex of 17-day-old control, Cdk5 cKO2, and dKO mice, were dissolved in T-PER buffer, and they were immunoprecipitated using 4 μg of anti-Cdk5 (C8) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) [21 (link)]. Immunoprecipitated proteins (IP) were washed three times in cold PBS, and twice in kinase buffer (20 mM Tris HCl (pH 7.4), 10 mM MgCl2, 1 mM EDTA). Then, the IP was mixed with the kinase assay mixture (100 mM Tris HCl (pH 7.4), 50 mM MgCl2, 5 mM EDTA, and 5 mM DTT) plus 5 units of (γP32)-ATP, and using 5 μg of histone H1 as a substrate. The kinase assays were carried out for 30 minutes at 30°C, and the kinase activity reaction was stopped by adding 5× sodium dodecyl sulfate sample buffer and boiling for ten minutes at 70°C. Kinase reaction was electrophoresed on a 4 to 20% polyacrylamide gel, and then gels were exposed to X-ray films, for 1 to 3 hours at -80°C. The incorporation of P32 to histone H1 was quantified to measure band intensity using Scion Image Alpha 4.0.3.2 software (Scion Corporation, Frederick, MD, USA).
3
CDK5 Kinase Assay Protocol
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CDK5 kinase assays were performed as previously described.28 (link) Briefly, homogenized brain samples were incubated overnight at 4 °C with the anti-CDK5 antibody (C8; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and the antibody-CDK5 complex was precipitated with the Protein A-Sepharose beads for 2 h at 4 °C. Immunoprecipitates were then washed three times with the lysis buffer, and then once with the kinase buffer containing 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 10 mM MgCl2, 10 mM sodium fluoride and 1 mM sodium orthovanadate. CDK5 kinase assays were performed in the same buffer containing 1 mM DTT, 0.1 mM ATP and 0.185 MBq of [γ-32P]ATP with 20 ng of histone H1 as a control substrate. The reaction was performed in a final volume of 50 μl, and was incubated for 60 min at 30 °C, stopped subsequently by addition of 10% SDS sample buffer followed by heating at 95 °C for 5 min. Twenty-five microliter aliquots of the reaction mixture were plotted on a Whatman p80 paper, washed, dried and were placed in a scintillation vial for counting.
4
Antibody Reagents for Protein Analysis
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The anti-myc antibody (4A6) was obtained from Millipore (Billerica, MA). The anti-CDK5 antibody (C-8) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-actin antibody was purchased from Sigma (St Louis, MO). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and HRP-conjugated swine anti-rabbit IgG were purchased from Dako (Glostrup, Denmark). Alexa Fluor 488-conjugated anti-mouse IgG was purchased from Invitrogen (Carlsbad, CA). 4′,6-Diamidino-2-phenylindole (DAPI) was from Dojindo (Kumamoto, Japan). Phos-tag acrylamide was obtained from Wako (Osaka, Japan). Protein A/G Sepharose was purchased from GE Healthcare (Buckinghamshire, UK).
5
Measuring Cdk5 Activity in Stressed Rat Brains
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Cdk5 activity was measured as described previously [41 (link)]. Briefly, 300 μg of protein were extracted from brain cortex of control and stressed rats. Proteins were dissolved in T-PER buffer and immunoprecipitated using 4 μg of anti-Cdk5 antibody C8 (Santa Cruz, CA). Immunoprecipitated proteins (IP) were washed 3 times in cold PBS, and 2 times in kinase buffer [20 mM Tris HCl (pH 7.4), 10 mM MgCl2 and 1 mM EDTA]. IP were then mixed with the kinase assay mixture [100 mM Tris • HCl (pH 7.4), 50 mM MgCl2, 5 mM EDTA, and 5 mM DTT] plus 5 μCi (γP32)-ATP, with 5 μg of Histone H1 used as a substrate. Kinase assays were carried out at 30°C for 30 min and the kinase activity reaction was stopped by adding 5xSDS sample buffer and boiling it for 10 min at 95°C. The kinase reaction was electrophoresed on a 12% polyacrylamide gel and then gels were exposed to X-ray films for 1–3 h at −80°C. The incorporation of P32 to Histone H1 was quantified to measure band intensity using ImageJ 1.46r software (NIH, MD).
6
Quantitative Western Blot Analysis of Alzheimer's Biomarkers
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SK-N-BE and HEK-293 cells were homogenized in lysis buffer (150 mM NaCl, 20 mM Tris-HCl, pH 7.4, 1% Triton X-100, protease inhibitor mixture). Samples were centrifuged at 12,000 g for 15 min at 4 °C and supernatants were collected.
Western blot assay was performed under standard conditions. In brief, equal amounts of proteins were analyzed by 12% SDS-PAGE at 100 V for 90 min and blotted onto nitrocellulose membrane Hybond-enhanced chemiluminescence (ECL; Amersham Biosciences, Buckinghamshire, UK) in a Bio-Rad Trans-blot apparatus at 350 mA for 3 hours. The blots were probed with anti-p35 (C-19, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-BACE1 (D10E5, Cell Signaling, Beverly, MA), anti-CDK5 antibody (C-8, Santa Cruz Biotechnology), anti-pAPP (Thr668) (Cell Signaling, Danvers, CA, USA), anti-APP (Millipore, Billerica, MA, USA) and anti GAPDH (Novus biologicals, Littleton, CO, USA) antibodies followed by an alkaline phosphatase-conjugated IgG secondary antibody. Blots were processed by an ECL Plus detection kit as instructed by the supplier (Amersham Biosciences). All experiments were performed at least three times, and the quantization was made using the ImageJ program (National Institute of Health, Bethesda, MD, USA).
Western blot assay was performed under standard conditions. In brief, equal amounts of proteins were analyzed by 12% SDS-PAGE at 100 V for 90 min and blotted onto nitrocellulose membrane Hybond-enhanced chemiluminescence (ECL; Amersham Biosciences, Buckinghamshire, UK) in a Bio-Rad Trans-blot apparatus at 350 mA for 3 hours. The blots were probed with anti-p35 (C-19, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-BACE1 (D10E5, Cell Signaling, Beverly, MA), anti-CDK5 antibody (C-8, Santa Cruz Biotechnology), anti-pAPP (Thr668) (Cell Signaling, Danvers, CA, USA), anti-APP (Millipore, Billerica, MA, USA) and anti GAPDH (Novus biologicals, Littleton, CO, USA) antibodies followed by an alkaline phosphatase-conjugated IgG secondary antibody. Blots were processed by an ECL Plus detection kit as instructed by the supplier (Amersham Biosciences). All experiments were performed at least three times, and the quantization was made using the ImageJ program (National Institute of Health, Bethesda, MD, USA).
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