Histone proteins were extracted by acid precipitation. Briefly, cells from a 6-well dish were scraped, washed with PBS and resuspended in extraction buffer (PBS, 0.5% Triton-X-100 (9002-93-1, Sigma-Aldrich), 5 mM sodium butyrate (B5887, Sigma-Aldrich)), and allowed to extract for 20 mins. The extract was centrifuged at 14,000× g for 20 mins and the supernatant was discarded. The pellet was resuspended in 0.2 N HCl and left at 4 °C overnight. The mixture was vortexed, and the pH was neutralized with 1 M Tris-HCl pH 8.0. Western blots were performed using typical laboratory procedures with the antibodies: anti-H3K9me3 (1:1000; ab8898, abcam), anti-H3K4me3 (1:1000; ab8580, abcam), anti-H3K4me1 (1:1000; ab8895, abcam), anti-H3K27me3 (1:1000; 07-449, Millipore), anti-H3ac (1:1000; 06-599, Millipore), anti-H4K12ac (1:1000; ab46983, abcam), anti-H4ac (1:1000; 06-886, Millipore), anti-H3K27ac (1:1000; ab4729, abcam), anti-H2AK119ub (1:1000; 8240, Cell Signaling Technology), anti-H3 (1:1000; ab1971, abcam), anti-OCT4 (1:10,000; SC-8628, Santa Cruz), anti-NANOG (1:1000; A300-397A, Bethyl), and anti-GAPDH (1:10,000; MAB374, Millipore). Uncropped raw images of the Western blot membranes are in Supplementary Figure 8 .
Sc 8628
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Sc-8628 is a laboratory equipment product offered by Santa Cruz Biotechnology. It is designed for general laboratory use. The core function of the Sc-8628 is to facilitate standard laboratory procedures and experiments. No further details about its intended use or specifications are provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab80892, by Abcam (3 mentions)
Sc 5279, by Santa Cruz Biotechnology (3 mentions)
Sc 9081, by Santa Cruz Biotechnology (3 mentions)
Ab13840, by Abcam (3 mentions)
Ab8895, by Abcam (2 mentions)
Ab1791, by Abcam (2 mentions)
A21202, by Thermo Fisher Scientific (2 mentions)
Ab17721, by Abcam (2 mentions)
A11055, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Ab21624, by Abcam (2 mentions)
Ab198579, by Abcam (2 mentions)
Mab17591, by R&D Systems (2 mentions)
Mab4419, by Merck Group (2 mentions)
Ic1759p, by R&D Systems (2 mentions)
Mu392a uc, by BioGenex (2 mentions)
Ab10812, by Abcam (2 mentions)
Sc 17320, by Santa Cruz Biotechnology (2 mentions)
Ab23345, by Abcam (2 mentions)
Sc 67249, by Santa Cruz Biotechnology (2 mentions)
30 protocols using sc 8628
1
Histone Extraction and Western Blot Analysis
2
Immunophenotyping of Stem Cell Cultures
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Cells were fixed with 4% PFA for 30 minutes and incubated in 1% Triton-X-100 for 15 minutes to permeate the cell membrane. Nonspecific binding was blocked with 1% BSA at room temperature for 1 hour. Proteins were detected with specific primary antibodies at 4 °C overnight. Primary antibodies were as follows: anti-NESTIN (1:100, MAB353; Millipore), anti-TuJ 1 (1:200, T2200; Sigma-Aldrich), anti-OCT4 (1:200, sc-8628; Santa Cruz), anti-NANOG (1:200, ab80892; Abcam), anti-phospho (Ser465/467)-SMAD2 (1:200, #3108; CST), and PAX6 (1:200, AB_528427; DSHB). After three washes with PBS, cells were incubated with corresponding secondary antibodies (1:1000; Jackson ImmunoResearch) for 1 hour. DNA was counterstained with Hoechst33342 (Invitrogen) for 5 minutes at room temperature. Immunofluorescent images were obtained on an Axioplan Zeiss microscope (LSM 780; Carl Zeiss). Quantitative analysis of immunofluorescent staining was performed using ImageJ software when the immunofluorescent images were obtained at the same exposure parameters.
For FACS analysis, cells were digested into single cells, followed by two washes in DPBS. The cells were then filtered through a 35-μm cell strainer cap (Falcon™ Cell Strainers, 352235). Sox1-GFP cells were sorted and counted by flow cytometry. Analysis was performed on a FACS-Canto flow cytometer (Beckman Coulter MoFlo™ XDP).
For FACS analysis, cells were digested into single cells, followed by two washes in DPBS. The cells were then filtered through a 35-μm cell strainer cap (Falcon™ Cell Strainers, 352235). Sox1-GFP cells were sorted and counted by flow cytometry. Analysis was performed on a FACS-Canto flow cytometer (Beckman Coulter MoFlo™ XDP).
3
Immunoprecipitation and Fractionation Protocol
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For immunoprecipitation, cells were lysed on ice in TNE buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.5% Tergitol-type NP-40, 1 mM EDTA, and protease inhibitors). Anti-FLAG M2 magnetic beads (M8823, Sigma) were incubated overnight with cell lysate fractions. Samples were then washed 6 times with TBS. Western blotting was performed following standard principles and immunofluorescence was assessed using a Leica TCS SP2 spectral confocal microscope. Nuclear-cytoplasmic fractionation was performed following standard protocol. The following primary antibodies were used: anti-FLAG (F7425, Sigma), anti-NCoR (ABE251, Millipore), anti-HA (H6908, Sigma), anti-SMRT (ab24551, Abcam), anti-HDAC7 (ab12174, Abcam), anti-E-cadherin (BD Biosciences), anti-SSEA-1 (MC480, Cell Signaling), anti-SOX2 (MAB2018, R&D Systems), anti-OCT4 (sc-8628, Santa Cruz), anti-KLF4 (AF3158, R&D Systems), anti-MYC (AF3696, R&D Systems), anti-histone H3 (ab1791, Abcam), anti-ACTIN (A5316, Sigma), and anti-GAPDH (G8795, Sigma). DAPI was purchased from Sigma (D9542).
4
Immunofluorescence Analysis of Embryonic Proteins
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Embryos (n = 38, 3 replicates) were fixed in 3.7% paraformaldehyde for 20 min at room temperature, permeabilized with PBS/PVA containing 0.5% Triton X-100 at 37 °C for 1 h, and then incubated in PBS/PVA containing 1.0% bovine serum albumin at 37 °C for 1 h. Subsequently, the embryos were incubated overnight at 4 °C with anti-LC3 (ab58610, 1:100; Abcam, Cambridge, UK), anti-cytochrome C (ab110325, 1:100; Abcam), anti-p53 (sc6243, 1:100; Santa Cruz Biotech, CA, USA) and anti-OCT4 (sc8628, 1:100; Santa Cruz Biotech) antibodies. To check the fluorescence signal of 5mC, blastocysts were denatured with 1 N HCl at room temperature for 30 min and neutralized with 0.1 M Tris-HCl, pH 8.0 for 15 min. Subsequently, blastocysts were incubated in PBS containing 1% BSA, and then incubated overnight at 4 °C with 5mC antibody (ab10805, 1:100, Abcam, Cambridge, UK). After washing three times with PBS/PVA, the oocytes and embryos were incubated at 37 °C for 1 h with either goat anti-rabbit IgG (A11011, 1:200, Invitrogen) or rabbit anti-goat IgG (A11079, 1:200, Invitrogen), anti-mouse IgG (A21202, 1:200, Invitrogen). The oocytes and embryos were then stained with Hoechst 33342 for 5 min, washed three times with PBS/PVA, mounted onto slides, and examined using a confocal microscope (Zeiss LSM 710 META, Jena, Germany). Images were processed using Zen software (version 8.0, Zeiss, Jena, Germany).
5
Stem Cell Characterization Protocol
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The cells were washed with phosphate buffered saline (PBS) and then fixed with 4% (w/v)
paraformaldehyde (PFA) for 15 min. Alkaline phosphatase (ALP) activity was tested using
Alkaline Phosphatase Kit (Sigma-Aldrich, MO, USA) following the manufacturer’s
instructions. The pink-to-red colored colonies were classified as positive to ALP
activity. To investigate protein expression, the cells were passaged onto a cover slip and
then fixed with 4% (w/v) PFA. The cells were permeabilized if necessary in mixture of 0.1%
Triton X-100, 2% bovine serum albumin (BSA) in PBS and the non-specific binding was
blocked with 2% BSA. The cells were incubated at 4°C with primary antibodies overnight.
The primary antibodies in this study included OCT-3/4 (SC8628, Santa Cruz Biotechnology,
TX, USA , 1:100), SSEA-4 (ab16287, Abcam, Cambridge, UK , 1:50), FLK1 (SC393163, Santa
Cruz Biotechnology, 1:100) and cTnT (troponin T, ab33589, Abcam, 1:100). The samples were
then stained with secondary antibody corresponding to the primary antibodies used. The 4’,
6’-diamidino-2-phenylindole (DAPI) in mounting medium (VECTASHIELD® Mounting
Medium, Vector Laboratories, CA, USA) was used to visualize the nucleus. The negative
control was performed as described above without primary antibody. A fluorescent
microscope (BX51, Olympus) and DP2-BSW software were used for visualization and record the
samples.
paraformaldehyde (PFA) for 15 min. Alkaline phosphatase (ALP) activity was tested using
Alkaline Phosphatase Kit (Sigma-Aldrich, MO, USA) following the manufacturer’s
instructions. The pink-to-red colored colonies were classified as positive to ALP
activity. To investigate protein expression, the cells were passaged onto a cover slip and
then fixed with 4% (w/v) PFA. The cells were permeabilized if necessary in mixture of 0.1%
Triton X-100, 2% bovine serum albumin (BSA) in PBS and the non-specific binding was
blocked with 2% BSA. The cells were incubated at 4°C with primary antibodies overnight.
The primary antibodies in this study included OCT-3/4 (SC8628, Santa Cruz Biotechnology,
TX, USA , 1:100), SSEA-4 (ab16287, Abcam, Cambridge, UK , 1:50), FLK1 (SC393163, Santa
Cruz Biotechnology, 1:100) and cTnT (troponin T, ab33589, Abcam, 1:100). The samples were
then stained with secondary antibody corresponding to the primary antibodies used. The 4’,
6’-diamidino-2-phenylindole (DAPI) in mounting medium (VECTASHIELD® Mounting
Medium, Vector Laboratories, CA, USA) was used to visualize the nucleus. The negative
control was performed as described above without primary antibody. A fluorescent
microscope (BX51, Olympus) and DP2-BSW software were used for visualization and record the
samples.
6
Co-IP and GST Pull-down Assays for Oct4 Interactions
7
Immunofluorescence Staining of Pluripotent Markers
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Cells were fixed with 4% PFA, permeabilized using 0.2% Triton X-100, and treated with Ultra Vision block (ThermoFisher). Primary antibodies were diluted in 0.1% Tween-20 PBS and incubated either overnight at 6 °C with the given dilutions or 2 days in 6 °C with halved primary antibody amounts. Secondary antibody incubations were done in room temperature for 30 min in the presence of Hoechst33342 to stain the nuclei. Primary antibodies used were: LIN28A (1:250, D84C11 and D1A1A, Cell Signaling), NANOG (1:250, D73G4, Cell Signaling), OCT4 (1:500, sc-8628, Santa Cruz), SOX2 (1:250, D6D9, Cell Signaling), KLF4 (1:250, HPA002926, Sigma-Aldrich), C-MYC (1:250, D3N8F, Cell Signaling; 1:250, [Y69] ab32072, Abcam), TRA-1-60 (1:50, MA1-023, ThermoFisher), TRA-1-81 (1:100, MA1-024, ThermoFisher) TUBB3 (1:500, MAB1195, R&D Systems), AFP (1:400, A0008, Dako), SMA (1:200, A2547, Sigma), VIMENTIN (1:500, sc-5565, Santa Cruz). SOX17 (1:500, AF1924, R&D Systems), acetyl Histone 3 (1:500, ab47915, Abcam). Secondary antibodies used were: AlexaFluor 488: donkey anti-goat (1:500, A11055 and 11058; Invitrogen), donkey anti-mouse (1:500, A21202 and A21203; Invitrogen) and donkey anti-rabbit (1:500, A21206 and A21207; Invitrogen).
8
Immunofluorescence Characterization of Stem Cells
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The pPSCs and the differentiated cells were fixed with 4% (w/v) paraformaldehyde (PFA) for 40 min at room temperature and were washed three times with phosphate-buffered saline (PBS, Sigma). Then, they were permeabilized with 1% (v/v) Triton X-100 in PBS overnight at 4°C and were blocked with 1% (w/v) BSA in PBS for 1 hour at 37°C. They were incubated with primary antibodies for OCT4 (Santa Cruz, Sc-8628, 1:50), SOX2 (Santa Cruz, sc-17320, 1:250), NANOG (Pepro Tech, 500-P236, 1:250), CDX2 (Biogenex, MU392A-UC, 1:50), AFP (Abnova, H00000174, 1:50), VIMENTIN (Sigma-Aldrich, V6630, 1:50) and β-TUBULIN (Sigma-Aldrich, T5201, 1:100) overnight at 4°C. After they were incubated, the cells were washed three times with 0.01% (v/v) Triton X-100 and 0.1% (v/v) Tween-20 in PBS at room temperature. The cells were then incubated with secondary antibodies (Alexa Fluor® 488 for donkey anti-goat, donkey anti-rabbit and donkey-anti mouse, Invitrogen) diluted at 1:500 in 0.01% (v/v) Triton X-100 and 0.1% (v/v) Tween-20 in PBS for 1 hour at 37°C. Then, the cells were washed three times. Cell nuclei were stained with Hoechst 33342 (Sigma) for 8 minutes and were subsequently washed three times. Finally, the stained cells were mounted on glass slides and examined using a Nikon 80i microscope.
9
Immunofluorescence analysis of OS-55 cell line
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FACS-sorted SP and non-SP cells from the OS-55 cell line were fixed in BD Cytofix solution (BD Biosciences) and incubated for 20 min at 4°C. Following blocking in donkey serum (Sigma-Aldrich) for 20 min, cells were incubated with goat anti-Oct3/4A polyclonal primary antibody (1:100; sc-8628; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight at 4°C, and were subsequently incubated with rhodamine red-conjugated donkey anti-goat antibody (1:200; 705-295-003; Jackson Immunoresearch Laboratories, Inc., West Grove, PA, USA). For CD44 and Nanog immunofluorescence analysis, cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-human Nanog (1:5; 674206; BioLegend, San Diego CA, USA) or CD44 (1:5; 8011-0441; eBioscience, Inc., San Diego, CA, USA) antibodies. Human embryonic stem cells were used as the positive control. Cells were observed under a confocal microscope (LSM 700; Zeiss AG, Oberkochen, Germany). Images were captured and processed by Adobe Photoshop CS4 (Microsoft Corporation, Redmond, WA, USA).
10
Alkaline Phosphatase and Pluripotency Marker Detection
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For alkaline phosphatase (AP) detection, the commercial Alkaline Phosphatase Detection Kit (86R-1KT, Sigma-Aldrich) was used. Briefly, colonies were fixed and analyzed according to the manufacturer’s instructions. Colonies stained in pink were considered positive. AP positive clonal lineages were further analyzed for the detection of NANOG, OCT4, and SOX2.
biPSCs colonies were washed with PBS, permeabilized with 0.1% Triton X for 20 min, washed with PBS, and blocked with 1% BSA/0.05% Tween for 1 h and labeled for 1 h with primary antibodies: NANOG (Ab21624, Abcam—1:100 dilution), OCT4 (Sc8628, Santa Cruz—1:100 dilution), and SOX2 (Ab97954, Abcam—1:250). Cells were then washed with 0.05% Tween, incubated for 1 h with a secondary antibody (anti-rabbit Alexa Fluor 488-1:500 dilution), washed as described above, and labeled with Hoechst 33342 (Sigma-Aldrich—1:1000 dilution) for 5 min. After a final wash with PBS, the cells were photo-documented.
biPSCs colonies were washed with PBS, permeabilized with 0.1% Triton X for 20 min, washed with PBS, and blocked with 1% BSA/0.05% Tween for 1 h and labeled for 1 h with primary antibodies: NANOG (Ab21624, Abcam—1:100 dilution), OCT4 (Sc8628, Santa Cruz—1:100 dilution), and SOX2 (Ab97954, Abcam—1:250). Cells were then washed with 0.05% Tween, incubated for 1 h with a secondary antibody (anti-rabbit Alexa Fluor 488-1:500 dilution), washed as described above, and labeled with Hoechst 33342 (Sigma-Aldrich—1:1000 dilution) for 5 min. After a final wash with PBS, the cells were photo-documented.
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