All IPs were performed as described before.12 (link),41 (link) Briefly, ESCs were expanded and cultured in 15-cm feeder-free gelatin-coated dishes for 1 day. Cells were washed and scraped in ice-cold PBS supplemented with Halt PIC (Thermo 78430). For crosslinked IPs, cells were incubated with 1% formaldehyde at room temperature for 8 mins, quenched with glycine (final concentration of 0.1 M), and washed with PBS before scrapping. Nuclear extracts were prepared and quantified with BSA assay (Thermo Scientific 23227). 1 mg of protein was incubated with 2 μg of antibody (anti-Flag CST, 2368; Rabbit-IgG CST, 3900) crosslinked to Protein G-conjugated magnetic beads (Dynabeads protein G, Invitrogen) overnight at 4°C. IPs were washed with buffer containing 20 mM HEPES, pH 7.6, 10% glycerol, 100 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA. Proteins were eluted by resuspending beads in Laemmli buffer and incubating them at 95°C for 5 mins and detected by Western blot as described above.
Halt pic
Manufactured by Thermo Fisher Scientific
Halt PIC is a solution designed to inhibit protease activity in protein samples. It is a concentrated formulation that can be added to samples to prevent degradation of proteins during processing and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Dynabeads protein g, by Thermo Fisher Scientific (2 mentions)
Bsa assay, by Thermo Fisher Scientific (2 mentions)
Mini protean electrophoresis chamber, by Bio-Rad (2 mentions)
Mini trans blot apparatus, by Bio-Rad (2 mentions)
Benzonase nuclease, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Gtx125888, by Abcam (1 mentions)
Srx 101a, by Konica Minolta (1 mentions)
Ab3479, by Abcam (1 mentions)
F1804, by Abcam (1 mentions)
Ab6276, by Abcam (1 mentions)
4 protocols using halt pic
1
Chromatin Immunoprecipitation Assay in ESCs
2
Co-immunoprecipitation of Tet1 in ESCs
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Co-immunoprecipitation experiments were performed as described before (29 (link),30 (link)). Briefly, ESCs were washed and collected in ice-cold PBS supplemented with Halt PIC (Thermo 78430). Nuclear extracts were prepared and treated with benzonase nuclease (Sigma, E1014), and incubated with 4 μg of antibody (anti-Tet1 (GeneTex, GTX125888), Rabbit-IgG (CST, 3900) crosslinked to Protein G-conjugated magnetic beads (Dynabeads protein G, Invitrogen) overnight at 4°C. IgG was used as control. Immunocomplexes were washed with buffer containing 20 mM HEPES, pH 7.6, 10% glycerol, 100 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA. The proteins were eluted in 2× Laemlli buffer at 95°C and analyzed by western blot as described above.
3
Western Blot Protein Detection Protocol
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Western blots were performed as before (29 (link),30 (link)). Briefly, 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 Halt PIC (Thermo, 78430) and PMSF (Sigma, 93482), lysates were quantified by BSA assay and resolved on 7–9% SDS-PAGE gel (Mini-PROTEAN electrophoresis chamber, Bio-Rad), and transferred to PVDF membranes (Mini Trans-Blot apparatus, Bio-Rad) in 5-10% methanol transfer buffer following manufacturer's protocols. Membranes were blocked in 5% milk in PBS with 0.1% Tween-20 (PBS-T) and incubated O/N at 4°C with primary antibodies (anti-Tet1 1:3000 (GeneTex, GTX125888), anti-Tet2 1:750 (Abcam, ab124297), anti-Lamin-b1 1:1000 (ABclonal, A1910), anti-Sin3a 1:1000 (Abcam, ab3479), anti-Ezh2 1:1000 (CST, 5246), anti-Chd4 1:1000 (Abcam, ab70469)). Secondary antibody incubations (HRP-anti-mouse, 401253, or HRP-anti-rabbit, 401393, Calbiochem 1:2500) were for 1 h at RT. Protein bands were identified with ECL chemiluminescence reagent (Amersham RPN2106). Lamin-b1 was used as loading control.
4
Western Blot Analysis of Epigenetic Regulators
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Western blots were performed as described before12 (link) using antibodies listed in Table S2 . Briefly, ESCs were cultured on feeder-free gelatin-coated plates for 1 day and protein was extracted using 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 Halt PIC (Thermo, 78430) and quantified with BSA assay (Thermo Scientific 23227). 20 μg of protein lysate was mixed with 2x Laemmli buffer and resolved on 7-9% SDS-PAGE gel (Mini-PROTEAN electrophoresis chamber, Bio-Rad), and transferred to PVDF membranes (Mini Trans-Blot apparatus, Bio-Rad) in 10% methanol transfer buffer following the manufacturer’s instructions. Membranes were blocked in 5% milk in PBS with 0.1% Tween-20 (PBS-T) and incubated overnight at 4°C with primary antibodies (anti-Tet2 1:1000 Abcam ab124297; anti-Tet1 1:3000 GeneTex, GTX125888; anti-Vinculin 1:1000 Proteintech 66305; anti-Flag 1:1000 Sigma M2 F1804; anti-Sin3a 1:1000 Abcam ab3479; anti-beta actin 1:30000 Abcam ab6276). Next day, membranes were washed twice with PBS-T (10min each) and incubated with secondary antibody (goat anti-mouse HRP 401253, or goat anti-rabbit HRP, 401393, Millipore 1:2500) for 1 hr at room temperature. Protein bands were detected using ECL chemiluminescence reagent (Amersham RPN2106) and standard radiography (Konica SRX-101A).
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