Immunoprecipitations were performed using Dynabeads protein A magnetic beads coupled to polyclonal GFP antibodies (ab290, Abcam, Cambridge, UK), which also recognize YFP and CFP, using the crosslinker, Bis(sulfosuccinimidyl)suberate (BS3), according to the manufacturer’s instructions (Thermo Fisher Scientific). Coupled beads were incubated with cleared lysates, under gentle rotation at 4 °C overnight, and further washed, three times, with 10 mM Tris-HCl, pH 7.5, 100 mM KCl before elution. Immunoprecipitated proteins were eluted in lithium dodecyl sulfate (LDS) loading buffer (Invitrogen, Thermo Fisher Scientific), containing 100 mM DTT, by heating the beads for 10 minutes at 70 °C, and separated briefly on a NuPAGE 3–8% Tris-Acetate protein gel (Invitrogen).
Dynabeads protein a
Manufactured by Abcam
Sourced in United Kingdom, United States
Dynabeads Protein A are superparamagnetic polymer beads coated with recombinant Protein A. Protein A has a high affinity for the Fc region of immunoglobulins, making these beads suitable for the isolation and purification of antibodies from biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti gfp antibody, by Abcam (3 mentions)
Anti myc, by Cell Signaling Technology (2 mentions)
Anti myc, by Abcam (2 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (2 mentions)
Ab290, by Abcam (1 mentions)
Nupage 3 8 tris acetate protein gel, by Thermo Fisher Scientific (1 mentions)
Lds loading buffer, by Thermo Fisher Scientific (1 mentions)
Nb100 142, by Novus Biologicals (1 mentions)
Glycine, by Merck Group (1 mentions)
Protease inhibitor cocktail tablet, by Merck Group (1 mentions)
Nextseq sequencer, by Illumina (1 mentions)
Hyper prep kit, by Roche (1 mentions)
Ab176840, by Abcam (1 mentions)
Smarter thruplex dna seq kit, by Takara Bio (1 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (1 mentions)
S220 focused ultrasonicator, by Covaris (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Ab8580, by Abcam (1 mentions)
Anti h3k36me3, by Abcam (1 mentions)
Bioruptor sonicator, by Diagenode (1 mentions)
10 protocols using dynabeads protein a
1
Immunoprecipitation of Fluorescent Proteins
2
MRE11 ChIP-seq and RPA ssDNA Sequencing
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MRE11 ChIP-seq (Paiano et al. 2020 (link)) and RPA single-strand DNA sequencing (Khil et al. 2012 (link); Tubbs et al. 2018 (link)) were performed as previously described. Twenty million pre-B cells were used per library for ChIP-seq and SSDS. Cells were fixed in 1% formaldehyde in culture media (Sigma F1635) for 10 min at 37°C, quenched with 125 mM glycine (Sigma), washed twice with cold 1× PBS, snap-frozen on dry ice, and stored at −80°C until sonication and ChIP. Sonication, immunoprecipatation, and library preparation were performed as previously detailed (Tubbs et al. 2018 (link); Paiano et al. 2020 (link)). Prior to all immunoprecipatations, sheared chromatin was precleared with 40 µL of Dynabeads Protein A (Thermo Fisher) for 30 min at 4°C. For MRE11 ChIP-seq, sheared chromatin was enriched with 4 µL of anti-MRE11 (Novus NB100-142) bound to Dynabeads Protein A overnight at 4°C. For RPA SSDS, sheared chromatin was enriched with 10 µg of anti-RPA32/RPA2 antibody (Abcam ab10359) on Dynabeads Protein A overnight at 4°C. During library preparation, kinetic enrichment of single-strand DNA was performed (Khil et al. 2012 (link)), thus sequencing only RPA-bound ssDNA, by heating sheared DNA fragments for 3 min at 95°C and allowing DNA to return to room temperature.
3
In vivo Co-immunoprecipitation of HEC2 and PIF4
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For in vivo co-immunoprecipitation (co-IP) assays, 35S:HEC2-GFP, 35S:mHEC2-GFP and 35S:PIF4-myc were crossed for each combination and double transgenic plants expressing both proteins were selected. Immunoprecipitation was conducted using 50 seedlings for each sample with Dynabeads protein A and anti-myc (Abcam, Cambridge, MA). Western blots using anti-myc (Cell Signaling Technologies, Danvers, MA) or anti-GFP antibody (Abcam, Cambridge, MA) were used to detect the proteins.
4
Co-immunoprecipitation of STARD10 Protein
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INS1 (832/13) cells were lysed in the following nondenaturing lysis buffer: 20 mM HEPES, 150 mM NaCl, 1% Igepal, protease inhibitors (Roche Diagnostics, complete, EDTA-free protease inhibitor cocktail tablets), and phosphatase inhibitors (Sigma, P5726). 6 μg of Rabbit IgG Isotype Control (Abcam, ab171870) or anti-PCTP-L (STARD10) antibody (Abcam. ab242109) was bound to 50 μL of Dynabeads Protein A for 1 h at 4 °C. For co-immunoprecipitation (Co-IP), 1 mg of protein lysate was incubated with the complex beads-antibodies overnight at 4 °C. Beads were then washed twice in lysis buffer and twice in PBS-Tween 0.01% prior to proteomic analysis by the Bristol Proteomics Facility.
5
ChIP-seq Analysis of Gametocyte Chromatin
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Two infected mice were used in each ChIP-seq experiment. ChIP-seq experiments were performed as previously described (Kaneko et al., 2015 (link)). The gametocyte-enriched blood was filtered using a Plasmodipur filter to remove white blood cells and fixed in 1% paraformaldehyde for 30 min with swirling. Red blood cells were lysed in 0.84% NH4Cl, and residual cells were lysed in a lysis buffer containing 1% SDS. The lysate was sonicated in Bioruptor 2. After centrifugation, the supernatant was diluted with dilution buffer, and chromatin was immunoprecipitated with Dynabeads protein A coated with an anti-GFP antibody (Abcam, ab290). DNA fragments were recovered from the precipitated chromatin and used for library preparation for sequencing. The library was prepared using a Hyper Prep Kit (KAPA Biosystems). Sequencing was performed on an Illumina NextSeq sequencer.
6
Chromatin Immunoprecipitation of Histone H3.3 Variants
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KNS-42 cells were fixed with 1% formaldehyde and incubated at room temperature for 15 min. The cells were collected with centrifugation and re-suspended in ChIP-lysis buffer (50 mM HEPES-KOH, pH7.5, 150 mM NaCl, 1 mM EDTA, 0.5% (v/v) sodium deoxycholate, 1% (v/v) NP-40, 0.1% (w/v) sodium dodecyl sulfate) containing 1 × protease inhibitor cocktail (Nacalai Tesque). The chromatin was sheared by sonication with Focused-ultrasonicator S220 (Covaris) using milliTUBE 1-ml AFA Fiber. For ChIP, anti-histone H3.3 rabbit monoclonal antibody (Abcam, ab176840) and anti-histone H3.3 G34V rabbit monoclonal antibody were used as primary antibodies with pre-washed Dynabeads Protein A (Dynal). The epitope of the former antibody lies between the 50th and C-terminal residues of human histone H3.3. The ChIP-ed DNA was purified using the DNeasy Blood and Tissue kit (Qiagen) with some modifications, and 10 ng of the DNA was used for library preparation with the SMARTer ThruPLEX DNA-seq Kit (Takara Bio) according to the manufacturer's instruction. Sequencing was performed using the HiSeq X Ten or NovaSeq 6000 at Macrogen Japan Corp. ChIP-seq reads were mapped to the reference human genome GRCh37 (hg19) and peak calling was performed using MACS242 (link).
7
Coexpression and Immunoprecipitation of Drosophila Wing Disc Proteins
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Coexpression of DAxud1-GFP and NELF-B-HA on the imaginal wing disc was achieved with driver nub>Gal4. In total, 150 wing discs were dissected on PBS 1X, then precipitated and resuspended in RIPA buffer. Protein extracts were immunoprecipitated with GFP antibody (Abcam ab290) with O.N incubation at 4°C, then isolated with Dynabeads® Protein-A. Western blot assay was performed in cells with INPUT (total protein), NB (non bound fraction, supernatant post Dynabeads isolation), and protein precipitated with Dynabeads. Page ruler 2166 (thermoscienti c) was used as a weight marker.
8
In vivo Co-immunoprecipitation of Proteins
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For in vivo co-immunoprecipitation (co-IP) assays, 35S:HEC2-GFP, 35S:mHEC2-GFP and 35S:PIF4-myc were crossed for each combination and double transgenic plants expressing both proteins were selected. Immunoprecipitation was conducted using 50 seedlings for each sample with Dynabeads protein A and anti-myc (Abcam, Cambridge, MA).
Western blots using anti-myc (Cell Signaling Technologies, Danvers, MA) or anti-GFP antibody (Abcam, Cambridge, MA) were used to detect the proteins.
Western blots using anti-myc (Cell Signaling Technologies, Danvers, MA) or anti-GFP antibody (Abcam, Cambridge, MA) were used to detect the proteins.
9
ChIP-qPCR for Viral Minichromosomes
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The chromatin immunoprecipitation (ChIP) protocol was based on the method described by Saleh et al. [51 (link)]. The anti-histone H3K9me2 (ab194680; Abcam, Cambridge, MA, USA) and anti-histone H3K4me3 (ab8580; Abcam) antibodies were bound to protein A dynabeads for 4 h at 4 °C.
Symptomatic, recovered, and superinfected tissues (1 g) were used for isolation of PepGMV minichromosomes (500 μL). protein A dynabeads (Thermo Scientific) were used for pre-clearing for at least 2 h at 4 °C. After pre-clearing the minichromosomes extract was incubated with the antibody-protein A dynabeads overnight at 4 °C. DNA was extracted using phenol-chloroform, followed by ethanol precipitation. Purified viral DNA was quantified by real-time qPCR.
Symptomatic, recovered, and superinfected tissues (1 g) were used for isolation of PepGMV minichromosomes (500 μL). protein A dynabeads (Thermo Scientific) were used for pre-clearing for at least 2 h at 4 °C. After pre-clearing the minichromosomes extract was incubated with the antibody-protein A dynabeads overnight at 4 °C. DNA was extracted using phenol-chloroform, followed by ethanol precipitation. Purified viral DNA was quantified by real-time qPCR.
10
Chromatin Immunoprecipitation and RNA-Seq
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