ChIP assays were performed from approximately 107 cells per experiment, according to previously described protocol with slight modifications32 ,33 . Briefly, cells were crosslinked with 1% formaldehyde for 10 min at room temperature and formaldehyde was quenched by addition of glycine to a final concentration of 0.125 M. Chromatin was sonicated to an average size of 0.5–2 kb, using Bioruptor (Diagenode). 50–75uL of protein G dynal beads (Invitrogen) were used to capture 3–5ug of antibody in phosphate citrate buffer pH=5.0 (2.4 mM citric acid, 5.16 mM Na2HPO4) for 30 min at 27C. Antibody bead complexes were rinsed 2x with PBS and added to sonicated chromatin and rotated at 4C overnight. 10% of chromatin was reserved as “input” DNA. Magnetic beads were washed and chromatin eluted, followed by reversal of the crosslinkings and DNA purification. Resultant ChIP DNA was dissolved in TE.
Protein g dynal beads
Manufactured by Thermo Fisher Scientific
Sourced in China, United States
Protein G Dynal Beads are magnetic beads coated with recombinant Protein G, a bacterial cell wall protein that binds to the Fc region of various immunoglobulins. These beads can be used for the affinity purification and isolation of antibodies and other Fc-containing proteins from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Bioruptor, by Diagenode (4 mentions)
Anti v5 antibody, by Bio-Rad (2 mentions)
Nebnext ultra 2 dna library prep kit for, by Illumina (1 mentions)
Benzonase nuclease, by Merck Group (1 mentions)
Hmgb1 antibody, by Abcam (1 mentions)
Anti rabbit igg antibody, by Merck Group (1 mentions)
Complete mini edta free protease inhibitor cocktail, by Roche (1 mentions)
Ab4729, by Abcam (1 mentions)
Model 100, by Thermo Fisher Scientific (1 mentions)
Chip it kit, by Active Motif (1 mentions)
Anti α tubulin, by Thermo Fisher Scientific (1 mentions)
Anti mono adp ribose binding reagent, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Actin, by Cell Signaling Technology (1 mentions)
Vinculin, by Cell Signaling Technology (1 mentions)
Hrp labeled secondary antibody, by GE Healthcare (1 mentions)
11 protocols using protein g dynal beads
1
ChIP Assay Protocol: Chromatin Immunoprecipitation
2
Chromatin Immunoprecipitation (ChIP) Protocol
3
BG4-DNA-G4 Immunoprecipitation Sequencing
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BG4-DNA-IP-seq, DNA immunoprecipitation with anti-BG4 antibody coupled with sequencing, was conducted following our previously published protocols [15 (link)]. Briefly, a total of 5 μg of fragmented genomic DNA was denatured and reassociated in the G4 stabilization buffer (40% PEG200, 10 mM Tris-HCl, pH = 7.5, with 150 mM KCl or NaCl). The reassociated DNA-G4s were specifically recognized by using anti-BG4-FLAG antibody in the IP incubation buffer (50 mM HEPES, 150 mM KCl, 1 mM MgCl2, 130 nM CaCl2, 1% BSA, 40% PEG200, Complete Mini, pH = 7.5), which was followed by incubation with an additional anti-FLAG antibody (D110005, BBI, Shanghai, China) and washed protein G Dynalbeads (10004D, Invitrogen, Carlsbad, CA, USA). Protein-G-bead-bound anti-BG4-DNA-G4s complexes were finally collected and washed three times with washing buffer (10 mM Tris-HCl, 150 mM KCl, 1% Tween20). The BG4-bound DNA-G4s were finally eluted using 200 μL elution buffer (0.1 M NaHCO3, 1% SDS) two times. Each experiment was biologically repeated. The BG4-DNA-IPed DNA or input DNA was recovered for library preparation. All libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (E7645S, NEB, Lpswich, USA), and sequenced using the Illumina platform, followed by data analyses.
4
ChIP-Seq Protocol for Chromatin Analysis
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ChIP assays were performed from approximately 107 cells per experiment, according to previously described protocol with slight modifications [17] (link). Briefly, cells were crosslinked with 1% formaldehyde for 10 min at room temperature and formaldehyde was quenched by addition of glycine to a final concentration of 0.125 M. Chromatin was sonicated to an average size of 0.5–2 kb, using Bioruptor (Diagenode). 50–75 μL of protein G dynal beads (Invitrogen) were used to capture 3–5 μg of antibody in phosphate citrate buffer pH = 5.0 (2.4 mM citric acid, 5.16 mM Na2HPO4) for 30 min at 27 °C. Antibody bead complexes were rinsed 2x with PBS and added to sonicated chromatin and rotated at 4 °C overnight. 10% of chromatin was reserved as “input” DNA. Magnetic beads were washed and chromatin eluted, followed by reversal of the crosslinkings and DNA purification. Resultant ChIP DNA was dissolved in TE. Results were verified with qPCR of 7 selected regions [17] (link).
5
Immunoprecipitation and Mass Spectrometry Analysis
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PC-3 and SKOV-3 cells were lysed in 50 mM Tri-HCl pH 8.0, 150 mM NaCl, 0.1% NP-40, 1 mM EDTA, 2 mM MgCl2 and complete™ Mini, EDTA-free Protease Inhibitor Cocktail (Roche, Basel, Switzerland) and incubated with 1.5:1000 (v/v) Benzonase®Nuclease (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 4 °C to eliminate nucleic acids from the lysates. Total protein was quantified using the Bradford method. In total, 40 μg of HMGB1 antibody (Abcam, ab 18256, Cambridge, UK), THOC5 antibody (Abcam ab86070) or anti-rabbit IgG antibody (Millipore, Co., Burlington, MA, USA) were crosslinked to 50 μL of Protein G-Dynal beads (Invitrogen, Waltham, MA, USA) as previously described [11 (link)]. For each IP, 2.5–3 mg of total protein were incubated with antibody-coupled beads for 4 h at 4 °C and beads were then washed four times with IPP150 buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl and 0.1% NP-40) and four times with 50 mM ammonium bicarbonate. On-bead digestion was carried out as previously described [12 (link)].
6
ChIP Assay Protocol: Chromatin Immunoprecipitation
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ChIP assays were performed from approximately 107 cells per experiment, according to previously described protocol with slight modifications32 ,33 . Briefly, cells were crosslinked with 1% formaldehyde for 10 min at room temperature and formaldehyde was quenched by addition of glycine to a final concentration of 0.125 M. Chromatin was sonicated to an average size of 0.5–2 kb, using Bioruptor (Diagenode). 50–75uL of protein G dynal beads (Invitrogen) were used to capture 3–5ug of antibody in phosphate citrate buffer pH=5.0 (2.4 mM citric acid, 5.16 mM Na2HPO4) for 30 min at 27C. Antibody bead complexes were rinsed 2x with PBS and added to sonicated chromatin and rotated at 4C overnight. 10% of chromatin was reserved as “input” DNA. Magnetic beads were washed and chromatin eluted, followed by reversal of the crosslinkings and DNA purification. Resultant ChIP DNA was dissolved in TE.
7
Mapping H3K27ac Chromatin Modifications
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Satellite cells from two steers immediately before and 2 days after induction of differentiation were cross-linked in 1% formaldehyde for 10 min and then lysed in lysis buffer from the ChIP-IT kit (Active Motif, Carlsbad, CA, USA). Cell nuclei were suspended in ChIP buffer from the ChIP-IT kit and then sheared on ice by 10 pulses of 20-s sonication using a sonic dismembrator Model 100 at setting 3 (ThermoFisher Scientific) to generate chromatin fragments of 200 to 1000 bp. To identify the genomic regions associated with H3K27ac modification, chromatin fragments were incubated with an anti-histone H3K27ac antibody (ab4729, abcam, Cambridge, MA, USA) at 4 °C overnight with gentle rocking. The H3K27ac antibody-chromatin complexes were separated from unbound chromatin fragments using protein G-Dynal beads (ThermoFisher Scientific). Chromatin fragments immunoprecipitated by the H3K27ac antibody and those before immunoprecipitation (i.e., input chromatin) were reverse cross-linked by incubating them at 65 °C for 4 h. DNA was extracted and purified using spin columns from the ChIP-IT kit.
8
Verification of α-Tubulin Mono-ADP-Ribosylation
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Mono-ADP-ribosylation (MAR) of α-tubulin was verified by immunoprecipitation as previously described (Hutin et al., 2018a (link)). In brief, mNSCs were harvested and the cell pellet was lysed in cell lysis buffer mNSCs (200 mM NaCl, 1% NP40, and 20 mM HEPES at pH 7.4, supplemented with protease inhibitor cocktail; Sigma). Lysates were clarified by centrifugation and the supernatant was incubated for 2 h with anti- α-tubulin (Sigma) antibody and protein G Dynal beads (Thermo Fisher). The bead-antibody-protein complex was then washed three times for 5 min in wash buffer (200 mM NaCl, 0.1% NP40, and 20 mM HEPES at pH 7.4, supplemented with protease inhibitor cocktail; Sigma). The proteins were then eluted from the beads with 2× Laemmli buffer and separated by SDS–PAGE and transferred to PVDF membranes. Membranes were probed with anti-mono-ADP-ribose-binding reagent (Millipore) followed by incubation with the appropriate secondary antibody. A list of all antibodies is provided in Table 2 .
COS-1 cells were transfected with 2 μg of EGFP, GFP-Tiparp, or GFP-TiparpH532A plasmids (MacPherson et al., 2013 (link)) per well of a six-well plate using Lipofectamine 2000 reagent according to the manufacturer’s instructions. The next morning, cells were harvested, protein extracts were made and the immunoprecipitation was conducted as described above.
COS-1 cells were transfected with 2 μg of EGFP, GFP-Tiparp, or GFP-TiparpH532A plasmids (MacPherson et al., 2013 (link)) per well of a six-well plate using Lipofectamine 2000 reagent according to the manufacturer’s instructions. The next morning, cells were harvested, protein extracts were made and the immunoprecipitation was conducted as described above.
9
Isolation of Tagged Neuronal Nuclei
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Antibody-adsorbed Dynal Protein-G beads (Invitrogen: 100-03D) were generated as described previously57 (link). Briefly, 300 µl of beads were bound to 1µg of anti-GFP (Invitrogen: G10362), anti-FLAG (Sigma: F7425) or anti-V5 antibody (Abdserotec: MCA1360) in 1xPBS-0.01%Tween-20.
10XUAS-unc84-smFP_FLAG and 10XUAS-unc84-smFP_V5 reporter lines were generated by phiC31-mediated transgenesis58 (link). Tdc-GAL459 (link) was used to drive expression of both tags in octopaminergic neurons and nuclei were purified by the INTACT procedure57 (link). Approximately 200 frozen heads were added to 20 ml homogenization buffer (10 mM β-glycerophosphate pH 7.0, 2 mM MgCl2, 0.5% NP-40) and passed over a Yamato continuous flow homogenizer, set at 100 rpm, 6 times. The homogenate was then successively filtered through first a 20 µm and then a 10 µm nylon filter (Partec: 040042325, 0400422314). 60 µl of Dynal Protein-G magnetic beads adsorbed to anti-GFP, anti-FLAG or anti-V5 antibody were then added to the filtered homogenate, which was subjected to constant agitation at 4 °C for 30 minutes. Bead-bound nuclei were captured on a magnet, washed 3x with homogenization buffer, filtered through a 10 µm nylon filter and analyzed by both light and fluorescence microscopy.
10XUAS-unc84-smFP_FLAG and 10XUAS-unc84-smFP_V5 reporter lines were generated by phiC31-mediated transgenesis58 (link). Tdc-GAL459 (link) was used to drive expression of both tags in octopaminergic neurons and nuclei were purified by the INTACT procedure57 (link). Approximately 200 frozen heads were added to 20 ml homogenization buffer (10 mM β-glycerophosphate pH 7.0, 2 mM MgCl2, 0.5% NP-40) and passed over a Yamato continuous flow homogenizer, set at 100 rpm, 6 times. The homogenate was then successively filtered through first a 20 µm and then a 10 µm nylon filter (Partec: 040042325, 0400422314). 60 µl of Dynal Protein-G magnetic beads adsorbed to anti-GFP, anti-FLAG or anti-V5 antibody were then added to the filtered homogenate, which was subjected to constant agitation at 4 °C for 30 minutes. Bead-bound nuclei were captured on a magnet, washed 3x with homogenization buffer, filtered through a 10 µm nylon filter and analyzed by both light and fluorescence microscopy.
10
Quantitative Immunoblotting of Signaling Pathways
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Immunoblotting of lysates from cells and frozen tumor fragments was performed as described [52 (link)]. Immunoprecipitation of p85 from cell lysates was performed using Dynal protein-G beads (Invitrogen) and p85 antibody (Abcam) as described [53 ]. Following SDS-PAGE, proteins were transferred to nitrocellulose membranes, and even protein loading was visually confirmed by Ponceau S staining. Blots were probed with antibodies against P-AKTS473, P-AKTT308, AKT, P-p70S6KT389, P-S6S240/244, P-IGF-1RβY1135/6/P-InsRβY1150/1, IGF-1Rβ, InsRβ, IRS-1, IRS-2, actin, vinculin (Cell Signaling), p85 (Abcam), or ER (Santa Cruz). HRP-labeled secondary antibodies (GE Healthcare) and ECL substrate (Pierce) were used for signal detection. Densitometry analysis of immunoblot film was performed using ImageJ software, and relative signal values were analyzed by ANOVA followed by Bonferroni multiple comparison-adjusted post-hoc test between groups.
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