Nuclear extracts of mouse PF cortex were prepared in 0.25 M sucrose buffer (sucrose 0.25 M, Tris 50 mM, EDTA 1 mM, imidazole 3 mM, pH 7.0 + proteases inhibitor cocktail). Samples were centrifuged 10 min at 250 g and nuclear fraction was resuspended in Laemmli buffer. All samples were sonicated before protein assay (BCA Pierce, Thermoscientific) and Western blotting was performed on 20 µg of protein lysates. Membranes were incubated overnight at 4°C with the primary antibodies; anti-HDAC2 1∶1500 (Abcam), anti-H3 1∶10000 (Millipore), anti-tubulin 1∶4000 (Sigma), anti-CREB and phospho-CREB 1∶1000 (Millipore). Washes with PBS-Tween (0.005%) were followed by incubation with secondary antibody (1∶10 000 anti-mouse or anti-rabbit IgG) (GE Healthcare) coupled to horseradish peroxidase and revealed by ECL. For quantification, the membranes were stripped and reincubated with an anti-tubulin or an anti-H3 antibody. Immunoreactive bands were quantified with an electrophoresis Gel Doc 2000 imaging system coupled to a Quantity one software (Bio-Rad).
Phospho creb
Manufactured by Merck Group
Sourced in United States, Germany
Phospho-CREB is a laboratory reagent used for the detection and quantification of phosphorylated CREB (cAMP Response Element Binding Protein) in cellular samples. It is a specific antibody that binds to the phosphorylated form of the CREB protein, enabling researchers to measure the activation of this transcription factor in various experimental conditions.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (3 mentions)
Immunobilon fl transfer membrane, by Merck Group (2 mentions)
10r g109a, by Merck Group (2 mentions)
Mab5432, by Merck Group (2 mentions)
Anti creb, by Merck Group (1 mentions)
Pierce bca, by Thermo Fisher Scientific (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Anti mouse or anti rabbit igg, by GE Healthcare (1 mentions)
Anti tubulin, by Merck Group (1 mentions)
Gel doc 2000 imaging system, by Bio-Rad (1 mentions)
Beclin 1 sc 11 427, by Santa Cruz Biotechnology (1 mentions)
Anti pp1, by Santa Cruz Biotechnology (1 mentions)
Darpp 32, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Anti ha, by Roche (1 mentions)
C fos, by Santa Cruz Biotechnology (1 mentions)
5 protocols using phospho creb
1
Nuclear Protein Extraction and Western Blot
2
Autophagy Markers in Neurodegeneration
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Streptozotocin (STZ, S0130), LC3 (for immunoblotting, #L8918), and p62 (#P0067) were purchased from Sigma (St. Louis, MO, USA). LC3 for immunostaining (#PM036) was purchased from MBL international (Woburn, MA, USA). Cathepsin D (sc-377124), ATP6E (sc-20946) and Beclin-1 (sc-11427) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). p-AKT (#2965), Rab7 (#9367) and beta-actin (#4970) were procured from Cell signaling (Danvers, MA, USA). Phospho-CREB (#05-807) and TrkB (#07-225) were purchased from Millipore (Billerica, MA, USA). BDNF (AB1779SP) and calbindin (AB1778) were purchased from Chemicon (Billerica, MA, USA).
3
CREB and SIRT1 Protein Expression in VTA
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Mice were sacrificed throughout the LD cycle (n=6 per group or genotype per ZT) and brains were rapidly extracted and VTA was micropunched, followed by homogenization and sonication. Protein lysate (10µg) were boiled then separated by SDS-PAGE and transferred to Immunobilon-FL transfer membrane (Millipore), as described previously. The following primary antibodies were used: β-actin (1:2000; Sigma A2228), GAPDH (1:1000; Fitzgerald 10R-G109a), CREB (1:1000; Millipore MAB5432), phospho-CREB (1:1000; Millipore 06-519), and SIRT1 (1:1000; Millipore 07-131) (Supplementary Methods ). Optical densities were quantified by NIH ImageJ software and normalized to the reference protein. Samples with values ±1.5 standard deviations from group means were excluded.
4
Western Blot Analysis of Neuronal Proteins
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Cell or human brain tissue lysates were prepared with protein extraction solution (Pro-Prep; Intron, SungNam, Korea) in accordance with the manufacturer’s guidelines. Proteins were subjected to SDS-PAGE and subsequently transferred to PVDF membrane (Bio-Rad, Hercules, CA, USA) and blocked with 5% skim milk in TTBS buffer. Blots were incubated for 16 h at 4 °C with primary antibodies to DARPP-32 (1:1000; Cell Signaling, Danvers, MA, USA), phospho-DARPP-34 (1:1000; Cell Signaling), CREB (1:1000; Millipore, Darmstadt, Germany), phospho-CREB (1:1000; Millipore), c-fos (1:100; Santa Cruz), anti-HA (1:5000; Roche, Branchburg, NJ, USA), anti-spectrin (1:1000; Enzo Life Sciences, Farmingdale, New York, USA), anti-PP1 (1:200; Santa Cruz) and β-actin (1:1000; Sigma). The blots were washed in TTBS buffer, incubated with secondary antibodies for 1 h at 23 °C and visualized using enhanced chemiluminescence reagents (Thermo, Waltham, Massachusetts, USA).
5
CREB and SIRT1 Protein Expression in VTA
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