Cell line chromatin was prepared as previously described (33 (link), 48 (link), 49 (link)). Embryonic chromatin was prepared using E18.5 wildtype CD1 embryos. 8–12 whole pancreata per prep were microdissected, flash frozen, sheared (22g needle), and solution crosslinked using 2% formaldehyde in PBS for 10 min at room temperature. Chromatin fragments were generated by sonication using the Diagenode Bioruper Pico and precleared using Protein-G Dynabeads (10004D; Invitrogen). ChIP was then performed using EZ-Magna ChIP A/G Kit (Millipore, #17–10086). Briefly, chromatin aliquots were incubated with A/G beads and anti-Ldb1 (ab96799; Abcam or sc11198x; Santa Cruz) or rabbit IgG (P120–101; Bethyl Laboratories) antibodies at 4°C overnight with rotation. Bound complexes were washed and crosslinks were reversed, DNA was purified. qPCR was performed on immunoprecipitated DNA using SYBR Green PCR master mix (Bio-Rad) and primers for Pdx1 (19 (link)) and Ngn3 (24 (link)) regulatory sequences (Supplemental Table 2 ). For cell line ChIP, enrichment of target control sequences in ChIP DNAs was normalized to inactive albumin control sequences (50 (link)) and calculated relative to rabbit IgG enrichment set as one-fold (ΔΔCt). Enrichment of target regions for in vivo ChIP was calculated as percent input (as described (51 (link))) and plotted relative to rabbit IgG enrichment (set to one-fold).
Ab96799
Manufactured by Abcam
Sourced in United States
Ab96799 is a lab equipment product. It is a tool designed for use in scientific research and laboratory settings. The core function of this product is to facilitate specific laboratory tasks, though detailed information about its intended use is not available.
Automatically generated - may contain errors
Lab products found in correlation
Sc11198x, by Santa Cruz Biotechnology (1 mentions)
Ez magna chip a g kit, by Merck Group (1 mentions)
P120 101, by Fortis Life Sciences (1 mentions)
Protein g dynabead, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Bio-Rad (1 mentions)
Ab150361, by Abcam (1 mentions)
Ab226346, by Abcam (1 mentions)
Ab6328, by Abcam (1 mentions)
Bradford assay reagent, by Bio-Rad (1 mentions)
Anti pstat3 y705, by Cell Signaling Technology (1 mentions)
T6199, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Ab9110, by Abcam (1 mentions)
Ecl luminescent solution, by Merck Group (1 mentions)
Ecl plus western blotting detection reagents, by Merck Group (1 mentions)
Ibright cl1000 imaging system, by Thermo Fisher Scientific (1 mentions)
Hrp goat anti rabbit igg h l, by ABclonal (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
5 protocols using ab96799
1
Chromatin Immunoprecipitation Protocol for Developmental Transcription Factors
2
Western Blot Protein Analysis
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For Western blots, whole-cell extracts were prepared using the radioimmunoprecipitation assay (RIPA) lysis buffer (LPS solution, Daejeon, Korea) comprised of 150 mM NaCl, 1% NP-40, 0.1% SDS, and 50 mM Tris (pH 7.4) containing 1 mM NaF, 1 mM Na3VO4, 1 mM ß-glycerophosphate, 2.5 mM sodium pyrophosphate, and protease inhibitor cocktail (Roche, Basel, Switzerland). Proteins were quantified using the Bradford assay reagent (Bio-Rad) according to the manufacturer’s instructions. Proteins (20–30 μg) were separated on 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting was performed as described above. The primary antibodies used were as follows: Anti-pSTAT3 (Y705) (1:500, 9145S; Cell Signaling Technology, Danvers, MA, USA), anti-STAT3 (1:1000, 4904S; Cell Signaling Technology), anti-fibronectin (1:500, ab6328; Abcam, Cambridge, UK), anti-integrin α5 (1:1000, ab150361; Abcam), anti-JAK1 (1:500, 50996S; Cell Signaling Technology), anti-JAK2 (1:1000, 3230S; Cell Signaling Technology), anti-GP130 (1:2000, ab226346; Abcam), anti-LDB1 (1:1000, ab96799; Abcam), anti-LMO2 (1:500, NB110-78626SS; Novus Biologicals, Littleton, CO, USA), and anti-α-tubulin (1:10,000, T6199; Sigma Aldrich). α-Tubulin was used as a loading control.
3
Protein Expression Analysis Protocol
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The collected cells were rinsed with PBS and lysed by RIPA buffer (Byeotime, China). Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes (CST, USA), blocked with milk, added with primary antibody and shaken at 4 °C overnight. The PVDF membrane was incubated with the secondary antibody at room temperature, and the ECL luminescent solution (Millipore, USA) was added to the membrane for visualization with AI600 image gel imaging analyzer (GE, USA). The following primary antibodies were used: LMO2 (ab91652, abcam, USA), LDB1 (ab96799, abcam, USA), HA (ab9110, abcam, USA), c-Myc (9402, CST, USA), PARP (9542, CST, USA), Flag (14793, CST, USA), GAPDH (5174, CST, USA) as a reference protein.
4
Western Blot Analysis of RGS5 Protein
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Total protein in HEMECs was separated by SDS-PAGE. The proteins were transferred onto polyvinylidene fluoride (PVDF) membranes from SDS-PAGE. Then incubated the PVDF membrane with rabbit anti-RGS5 (ab96799, 1: 1500, Abcam, Cambridge, MA) primary antibodies at 4°C for 12 h-16 h, then incubated at room temperature for 2 hours with secondary antibody. RGS5 protein was visualized using ECL Plus Western blotting Detection Reagents (Millipore, Billerica, MA).
5
Western Blot Analysis of LDB1, Pygo2, and H3
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HEK293T Cells were lysed with RIPA buffer (Beyotime) supplemented with 1 mM PMSF (Sigma) and incubated for 5 min on ice, followed by 21,130 g centrifugation for 10 min. Supernatants were transferred to new tubes, protein concentrations were determined using the BCA protein assay kit (Beyotime), and 20 μg of total protein in SDS loading buffer were analyzed by SDS-PAGE. Proteins were transferred to PVDF membranes (Millipore) using the Trans-Blot Turbo Transfer system (Bio-Rad). Membranes were blocked in 5% (w/v) milk (BD) in buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% Tween-20, probed at 4 °C overnight with primary antibody: rabbit polyclonal LDB1 antibody (Abcam, AB96799, 1:1000 dilution), rabbit polyclonal Pygo2 antibody (Proteintech, 11555-1-AP, 1:600 dilution) or rabbit polyclonal histone 3 antibody (Proteintech, 17168-1-AP, 1:5000 dilution), respectively, followed by incubating for 1 h at room temperature with HRP goat anti-Rabbit IgG (H + L) (Abclonal, AS014, 1:5000 dilution). Blots were developed with Clarity Western ECL Substrate (Bio-Rad) and exposed with iBright CL1000 imaging system (Invitrogen).
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