ALP staining was performed using the Alkaline Phosphatase Detection Kit (Chemicon) as instructed by the manufacturer. Immunocytochemistry was performed using standard protocol. Briefly, cells were fixed in 2% paraformaldehyde (Sigma-Aldrich), washed three times with PBS, and then incubated in PBS containing 0.1% Triton X-100 and 3% skim milk in PBS for 1 h at room temperature. The cells were then incubated with the following primary antibodies at 4°C overnight: Nanog (ab21603, 1:500, Abcam); Oct4 (sc-5279, 1:100, Santa Cruz); Sox2 (AB5603, 1:500, Millipore); SSEA1 (sc-21702, 1:100, Santa Cruz); Tuj-1 (MMS-435P, 1:500, Covance); CD31 (sc-1506-R, 1:100, Santa Cruz); and IgG (sc-2025, 1:500, Santa Cruz). After washing three times with PBS, cells were incubated with secondary antibodies: Alexa Fluor 488 goat anti-mouse IgG (1:1000, Molecular Probes) and Alexa Fluor 488 goat anti-rabbit IgG (1:1000, Molecular Probes) for 2 h at room temperature. Nuclei were detected by propidium iodide (PI) (Vector Laboratories, Inc.) staining. Images were analysed by confocal microscopy (FV1000; Olympus).
Oct4 sc 5279
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Oct4 (sc-5279) is a lab equipment product manufactured by Santa Cruz Biotechnology. It is a protein that functions as a transcription factor and plays a crucial role in the self-renewal and pluripotency of embryonic stem cells.
Automatically generated - may contain errors
Lab products found in correlation
Nanog a300 397a, by Fortis Life Sciences (2 mentions)
Rinf 84546s, by Fortis Life Sciences (2 mentions)
Tet2 ab124297, by Abcam (2 mentions)
Flag 14739s, by Abcam (2 mentions)
Mini protean electrophoresis chamber, by Bio-Rad (2 mentions)
Mini trans blot apparatus, by Bio-Rad (2 mentions)
Actin ac 15, by Abcam (2 mentions)
H3 ab1791, by Abcam (2 mentions)
Propidium iodide, by Vector Laboratories (1 mentions)
Cd31 sc 1506 r, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Fv1000, by Olympus (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Alkaline phosphatase detection kit, by Merck Group (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Tuj1 mms 435p, by Fortrea (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
H3k14ac, by Cell Signaling Technology (1 mentions)
Phospho acc, by Cell Signaling Technology (1 mentions)
11 protocols using oct4 sc 5279
1
Immunocytochemical Analysis of Stem Cell Markers
2
Protein Extraction and Western Blot Analysis
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Cell pellets were resuspended in RIPA buffer and were subjected to sonification. Protein concentration was determined using a bicinchoninic acid protein assay kit (Thermo Scientific, Rockford, IL, USA). Each well was loaded with 20 μg of proteins. Proteins were transferred to PVDF membrane (Bio-Rad). Antibodies for western blotting are as followed; DNMT3B (sc-20704) and OCT4 (sc-5279) from Santa Cruz (Dallas, TX, USA); ACTB (A5316) from Sigma.
3
Western Blot Analysis of Protein Markers
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Cells were harvested for the isolation of protein for western blot analysis using lysis buffer containing 1% NP40 detergent, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate (Sigma-Aldrich) and protease inhibitors (Roche, Mannheim, Germany). Protein concentrations were estimated using Bradford reagent, and an equal amount of protein (100 μg) was resolved by SDS-PAGE using Bio-Rad apparatus, transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA) and probed with appropriate antibodies. α-Tubulin (Calbiochem) served as loading control. Horseradish peroxidase-coupled secondary antibodies were obtained from The Jackson Laboratory, and immunoblots were visualized using PICO reagent (Pierce, Waltham, MA, USA). Primary antibodies used were phospho AMPK (Thr172; #2535), total AMPK α2 (#2757), AMPK α(1/2) (#2535), phospho ACC (#11818), total ACC (recognizes both isoforms 1 and 2; #3676), Bmi1 (#6964), CD44 (#37259), H3K14Ac (#MA5-24668) and H3 (#PA5-16183), which were obtained from Cell Signaling Technology (Beverly, MA, USA), and Nanog (sc-293121) and Oct-4 (sc-5279), which were obtained from Santa Cruz Biotechnology. All primary antibody dilutions were 1:1000 and secondary antibody dilutions were 1:5000.
4
Western Blot Analysis of Pluripotency Markers
5
Immunocytochemistry for Neural Stem Cells
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Cells were fixed in 4% of paraformaldehyde in Phosphate Buffer Saline (PBS), blocked for 30 min in PBS supplemented with 5% of normal donkey serum and 0.3% of triton X-100. Primary antibodies used were directed against FoxA2 (sc-6554, 1/100, Santa Cruz,); Ki67 (550609, 1/250, BD); Lmx1a (AB10533, 1/250, Millipore); Map2 (M2320, 1/500, Sigma); Nanog (AF1997, 1/250, R&F), Nestin (NB100-1604, 1/500, Novus Biologicals); Oct4 (sc-5279, 1/500, Santa Cruz); Pax6 (AB-528427, 1/1000, DSHB); Sox1 (AF3369, 1/500, R&D); TH (CH23006, 1/500, Acris Antibodies GmbH); Tra-1-81 (MAB4381, 1/500, Millipore); or βIII tub (MMS-435P, 1/1500, Covance). Nuclei were counterstained with Dapi (1/1500). Immunocytochemistry preparations were imaged using a Nikon A1 confocal microscope.
6
Isolation and Analysis of Oct4-Binding Proteins
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A vector containing mouse Oct4 promoter was constructed. The DNA vector was treated with the appropriate restriction enzyme, and only the mouse Oct4 promoter was purified. The purified DNA was methylated artificially with CpG Methyltransferase (M.SssI;(M0226S)(New England Biolabs, UK)), and biotinylated with biotin-labeled dCTP and Klenow enzyme. Biotinylated DNA was attached to streptavidin with Dynabeads kilobaseBINDER kit (60101)(Invitrogen, USA). The DNA was then incubated with the mESC protein extract. To remove the unbound proteins, beads were washed with washing buffer and magnets. Magnets pull streptavidin-Dynabeads, which were attached to biotinylated DNA with bound proteins. The last step was the elution of the proteins from the DNA and the protein analysis. After treatment with DNase (M6101)(Promega, USA) to eliminate DNA, we carefully took the supernatant. Finally, eluted proteins were separated using SDS-PAGE gel electrophoresis, and analyzed with MALDI-TOF and western blotting with Oct4 (sc-5279)(Santa Cruz, USA) and MBD2 (ab38646) (Abcam, UK) antibodies.
7
Western Blot Analysis of Pluripotency Markers
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For western blots, cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 250 mM NaCl, 2% Nonidet-P40, 2.5 mM EDTA, 0.1% SDS,0.5% DOC) supplemented with PIC and PMSF, resolved on 6%–10% SDS-PAGE (Mini-PROTEAN electrophoresis chamber, Bio-Rad), and transferred on PVDF membranes (Mini Trans-Blot apparatus, Bio-Rad) following manufacturer’s protocols. Membranes were blocked in 5% milk in PBS with 0.1% tween (PBST) and incubated overnight at 4°C, or for 1hr at room temperature with primary antibodies (Rinf 84546S, CST 1:1000; Nanog A300–397A, Bethyl Laboratories 1:2000; Oct4 SC-5279, SantaCruz 1:500; H3 ab1791, abcam 1:15000; Tet2 ab124297, abcam 1:1000; Flag 14739S, CST 1:1000; Actin AC-15, abcam 1:40000). Secondary antibody incubations (HRP-anti mouse, 401253, or anti rabbit, 401393, 1:5000, CalBiochem) were carried out for 1hr at room temperature. For quantifying Rinf protein levels in chromatin bound and soluble fractions of the cell, ESC lysate was fractionated as described before (Zhang et al., 2016 (link)). Each fraction was analyzed by western blot using anti-Rinf antibody. In all experiments Actin or H3 were used as loading controls.
8
Antibody-Based Protein Expression Analysis
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The antibodies were as follows: Brd4 (A301-985A50, Bethyl), H3 general (ab1791, Abcam, Lot # GR177884-2), H3.3 (09-838, Millipore, Lot # 2578126), H3K4me3 (39159, Active Motif), Spike-In antibody (61686, Active Motif, Lot# 00419007), OCT4 (sc-5279, Santa Cruz), NANOG (ab70482, Abcam), KLF4 (ab34814, Abcam), KLF5 (61099, Active Motif), SOX2 (AF2018, R&D Systems), Flag (2368, Cell Signaling), β-tubulin (T5201, Sigma), anti-mouse IgG-HRP (NA93V, GE, Lot # 9773218), anti-rabbit IgG-HRP (170-6515, Biorad, Lot # 350003248).
9
Immunofluorescence Staining of Stem Cell Markers
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The cells were fixed in 4% paraformaldehyde for 30 min and then were washed twice with PBS. After incubated for 2–3 h in blocking buffer containing 5% BSA and 0.2% Triton X-100, the cells were probed with the primary antibodies overnight at 4 °C. After washed three times with PBS, cells were incubated in blocking buffer containing a specific fluorescent secondary antibody and Hoechst 33342 (H3570, Invitrogen, 1:10,000) at 37 °C for 1 h. The cells were photographed under a Leica DMI8 microscope. The primary antibodies are Oct4 (SC-5279, Santa Cruz, USA, 1:500), Nanog (14295-1-AP, Proteintech, 1:500), Klf4(381633, ZENBIO, China, 1:500), and Sox2 (66411-1-Ig, Proteintech, 1:500).
10
Immunofluorescence Characterization of Cultured Cells
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Cultured cells were fixed in ice-cold 4% paraformaldehyde solution for 1 h, blocked in 2% skim milk (232100, Becton Dickinson) for 24 h at 4°C and incubated with primary antibodies for 24 h at 4°C.
The following primary antibodies were used: Flk1 (AVAS12, prepared in house, 1:500,000), PDGFRα (3174S, Cell Signaling Technology, 1:500), Oct4 (sc-5279, Santa Cruz Biotechnology, 1:200) and Nanog (RCAB002P, ReproCELL, Japan, 1:300).
After the primary antibody incubation, the cells were washed with phosphate buffered saline (PBS) three times and incubated with secondary antibodies conjugated to Alexa Fluor (Thermo Fisher Scientific, 1:500) for 24 h at 4°C. Nuclei were visualized with DAPI (4,6 diamidino-2-phenylindole; D3571, Thermo Fisher Scientific).
The following primary antibodies were used: Flk1 (AVAS12, prepared in house, 1:500,000), PDGFRα (3174S, Cell Signaling Technology, 1:500), Oct4 (sc-5279, Santa Cruz Biotechnology, 1:200) and Nanog (RCAB002P, ReproCELL, Japan, 1:300).
After the primary antibody incubation, the cells were washed with phosphate buffered saline (PBS) three times and incubated with secondary antibodies conjugated to Alexa Fluor (Thermo Fisher Scientific, 1:500) for 24 h at 4°C. Nuclei were visualized with DAPI (4,6 diamidino-2-phenylindole; D3571, Thermo Fisher Scientific).
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