Rabbit polyclonal antibodies were used to detect ERdj3, EDEM1, and ERp57 (Proteintech, Chicago, IL); others were to detect AAT (DAKO, Carpentaria, CA), GAPDH (Santa Cruz, Dallas, TX), Lamp1, P62, LC3B (all from Cell Signaling, Danvers, MA), and calnexin and calreticulin (both from Stressgen biotechnologies, San Diego, CA). Bafilomycin A1, MG132, Brefeldin A, and mouse monoclonal anti‐β‐actin were purchased from Sigma (St. Louis, MO), and 2C1 against human AAT polymers was purchased from Hycult biotech (Netherlands). Mouse monoclonal antibodies against BiP and AAT were purchased from BD bioscience (San Jose, CA) and R&D systems (Minneapolis, MN), respectively. Alexa Fluor 488 goat anti‐mouse IgG and Alexa Fluor 594 goat anti‐rabbit IgG were purchased from Invitrogen. Primers, Dynabeads protein A and G, Superscript VILO cDNA synthesis kit, and ERdj3 siRNA were purchased from Life Technologies, and HP DNA transfection reagent and TaqMan Universal PCR Master Mix were purchased from Roche Applied Science. Disuccinimidyl suberate (DSS) cross‐linker was purchased from Thermo Fisher scientific (Waltham, MA).
Protein a and g dynabeads
Manufactured by Thermo Fisher Scientific
Protein A and G Dynabeads are magnetic beads coated with recombinant forms of Protein A or Protein G, respectively. These beads are designed for the rapid and efficient isolation and purification of antibodies from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 4000, by Illumina (2 mentions)
Nebnext ultra 2 dna library prep kit, by Illumina (2 mentions)
Hiseq 2000 sequencer, by Illumina (2 mentions)
H3k27me3, by Merck Group (2 mentions)
Ab4729, by Abcam (2 mentions)
Hp dna transfection reagent, by Roche (1 mentions)
Mg132, by Merck Group (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Taqman universal pcr master mix, by Roche (1 mentions)
Lamp1, by Cell Signaling Technology (1 mentions)
Mouse monoclonal anti β actin, by Merck Group (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Disuccinimidyl suberate dss crosslinker, by Thermo Fisher Scientific (1 mentions)
Edem1, by Proteintech (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Calnexin, by Stressgen (1 mentions)
Calreticulin, by Stressgen (1 mentions)
13 protocols using protein a and g dynabeads
1
Antibody-Based Detection of ER Stress Proteins
2
Chromatin Immunoprecipitation (ChIP) Assay
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In vitro–cultured cells were cross-linked by 1% paraformaldehyde for 10 min at room temperature with gently shaking and were stopped by 100 mM glycine. The ChIP experiment was performed following the instructions of ChIP-IT Express ChIP kits (Active Motif). Antibodies, including anti-rabbit IgG [2729, Cell Signaling Technology (CST)], anti-HA (3724, CST), anti-H3K4me3 (07-473, Millipore), anti-H3K27me3 (07-499, Millipore), anti-H3K27Ac (39133, Active Motif), anti-H3K4me1 (39297, Active Motif), and Dynabeads protein A and G (Life Technologies) were used for IPs. The precipitated DNA was quantified by real-time PCR and normalized on the basis of total input DNA.
3
Chromatin Immunoprecipitation of Androgen Receptor
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Chromatin was immunoprecipitated from LNCaP cells treated with either 1 nM R1881 or vehicle (ethanol) for 1 h as described in Massie and Mills (2009) (link) using 10 µg AR n-20 antibody (sc-816; Santa Cruz Biotechnology) and an equal mixture of Dynabeads Protein A and G (10001D and 10003D; Life Technologies). Primer sequences are shown in Supplementary Table 4. Results are shown relative to the vehicle control. P-values were calculated using Wilcoxon rank-sum test.
4
ChIP-MS of NRPD1 Protein Complex
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ChIP-MS was performed with inflorescences from WT (Col-0), pNRPD1::NRPD1-3xFLAG, and pNRPD1::NRPD1-3xHA following the procedure described in the published protocol (21 (link)). Anti-FLAG M2 magnetic beads (Sigma-Aldrich, M8823) and HA antibody (Sigma-Aldrich, H6908) coupled with Dynabeads Protein A and G (Protein A:G ratio is 1:1) (Invitrogen, 10002 and 10004) were used in this assay to pull down the NRPD1 protein complex.
5
Immunoprecipitation Protocol for Protein Complexes
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Approximately 0.5 g of inflorescence was collected from each genotype and ground in liquid nitrogen into a fine powder, which was then resuspended in 2 ml of lysis buffer [50 mM tris (pH 7.5), 150 mM NaCl, 5 mM MgCl2, 10% glycerol, and 0.1% NP-40] containing protease cocktail inhibitors (MilliporeSigma, 4693132001). The lysate was cleared by centrifugation at 16,000g for 10 min at 4°C. The supernatants were incubated with 5 μl of anti-HA antibody (Sigma-Aldrich, H6908) and 30 μl of Dynabeads Protein A and G (protein A:G ratio is 1:1) (Invitrogen, 10002 and 10004) or with 10 μl of green fluorescent protein (GFP)–Trap (ChromoTek, gtma-20) at 4°C for 2 hours, under slow rotation. The beads were then washed five times for 5 min each with 1 ml of lysis buffer and resuspended in 50 μl of SDS-PAGE loading buffer. Input (15 μl) and bead eluate were used for Western blot analysis.
6
ChIP-seq for CTCF and Cp190 Binding Sites
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ChIP was performed with 2 μl of rabbit polyclonal antibody crude sera against CTCF1-293 or Cp1901-1096 (7 (link)) each incubated with half of the chromatin prepared from a biological replicate overnight at 4°C. Premixed Protein A and G Dynabeads (25 μl; Thermo Fisher Scientific, 100-01D and 100-03D) were added for 3 hours at 4°C and then washed for 10 min each once with RIPA, four times with RIPA with 500 mM NaCl, once in LiCl buffer [10 mM tris-HCl (pH 8), 250 mM LiCl, 1 mM EDTA, 0.5% IGEPAL CA-630, and 0.5% sodium deoxycholate], and twice in TE buffer [10 mM tris-HCl (pH 8) and 1 mM EDTA]. DNA was purified by ribonuclease digestion, proteinase K digestion, reversal of cross-links at 65°C for 6 hours, and elution from a QIAGEN MinElute PCR (polymerase chain reaction) purification column. ChIP-seq libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina. An equimolar pool of multiplexed ChIP-seq libraries at 4 nM was sequenced on two lanes of an Illumina HiSeq 4000 150-bp paired-end.
7
ChIP-Seq of CTCF and Cp190 Binding
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ChIP was performed with 2 µl of rabbit polyclonal antibody crude sera against CTCF1–293 or Cp1901–1096, each incubated with half of the chromatin prepared from a biological replicate overnight at 4 °C. Twenty-five microliters of pre-mixed Protein A and G Dynabeads (Thermo Fisher 100-01D and 100-03D) were added for 3 h at 4 °C, then washed for 10 min each once with RIPA, four times with RIPA with 500 mM NaCl, once in LiCl buffer (10 mM Tris-HCl pH 8, 250 mM LiCl, 1 mM EDTA, 0.5% Igepal CA-630, 0.5% sodium deoxycholate) and twice in TE buffer (10 mM Tris-HCl pH 8, 1 mM EDTA). DNA was purified by RNase digestion, proteinase K digestion, reversal of crosslinks at 65 °C for 6 h, and elution from a QIAGEN Minelute PCR purification column. ChIP-seq libraries were prepared using the NEBNext Ultra II DNA Library Prep kit for Illumina. An equimolar pool of multiplexed ChIP-seq libraries at 4 nM was sequenced on the Illumina HiSeq4000 (150 bp paired-end).
8
Chromatin Immunoprecipitation Sequencing of H3K27ac
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Lgr5EGFPhi cells were collected as for ChIP-PCR. ChIP-seq was performed as in.29 (link) Briefly, cross-linked cells were lysed and sonicated in RIPA lysis buffer to obtain 200- to 800-bp chromatin fragments. Chromatin were incubated overnight at 4°C with H3K27ac antibody followed by Protein A and G Dynabeads (10002D and 10004D) (Thermo Fisher Scientific) at 2 hours. Chromatin-antibody complex bound beads were washed twice in the sonication buffer, once in high-salt buffer, once in LiCl buffer, and once in Tris-EDTA, pH 8. Cross-links were reversed overnight by incubation at 65°C followed by treatment with Proteinase K (25530049; Thermo Fisher Scientific) for 1 hour at 55°C. DNA was purified with MinElute PCR purification kit (28004; Qiagen). Libraries were prepared using ThruPLEX DNA-seq kit (R400427; Rubicon Genomics, Ann Arbor, MI), purified using Ampure XP beads (Beckman Coulter, A63881) and sequenced on Illumina HiSeq X (Illumina, San Diego, CA) to obtain 150-bp paired-end reads.
9
CREB Binding to ETV5 and FBXW9 Promoters
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Chromatin immunoprecipitation (ChIP) was performed using Protein A and G Dynabeads (Thermo Fisher Scientific). For each ChIP reaction, 5 × 106 GSCs were cross-linked with 1% formaldehyde for 10 min at 37 °C, and genomic DNA was fragmented into ~100-to-300-bp pieces by sonication (truChIP Chromatin Shearing Reagent Kit, Covaris). DNA-bound CREB was immunoprecipitated using a CREB-specific antibody (Cell Signaling Technology). The associated DNA was then purified and analyzed by RT-qPCR to detect specific DNA sequences within the ETV5 or FBXW9 promoter bound by the CREB protein. The data were normalized to the input levels, and at least three independent biological replicates of each ChIP-qPCR were performed. An antibody against IgG (Abcam) was used as a nonspecific control. The primers were as follows: ETV5 prom 1, sense 5′- GACCTGAGGGGGAAGCTTAG-3′ and antisense 5′-TTTGCTGGATGGAGAAGTGG-3′; FBXW9 TSS, sense 5′-GCCCTAGGGGAAGCTCCATT-3′ and antisense 5′-AAAAGCGCAGAAACAGGAAC-3′.
10
ChIP-seq Protocol for Histone Modifications and Transcription Factors
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ChIP-seq was performed as previously described (60 (link)). Briefly, chromatin samples prepared from appropriate numbers of
fixed cells (2 × 105 for H3K27ac and
1 × 107 for CTCF, SMC1 and Pol II)
were sonicated and subsequently immunoprecipitated with individual antibodies
recognizing CTCF (Cell Signaling Technology), SMC1 (Bethyl lab), Phospho-Rpb1
CTD (Cell Signaling Technology), and H3K27ac (Abcam). Antibody-chromatin
complexes were captured with protein A and G Dynabeads (Thermo Fisher
Scientific) and washed with low salt wash buffer, high salt wash
buffer, and LiCl wash buffer. Chromatin-antibody immobilized on magnetic
beads were then subjected to tagmentation. Eluted DNA was purified using SPRI
Ampure XP beads (Beckman Coulter) and amplified for 8–12 cycles using
KAPA HiFi HotStart Ready mix (KAPA biosystems) and Nextera PCR primers
(Illumina). Libraries were purified using dual (0.65–0.25×) SPRI
Ampure XP beads (Beckman Coulter) and paired-end sequenced (100 bp) on the
Illumina HiSeq2500 platform.
fixed cells (2 × 105 for H3K27ac and
1 × 107 for CTCF, SMC1 and Pol II)
were sonicated and subsequently immunoprecipitated with individual antibodies
recognizing CTCF (Cell Signaling Technology), SMC1 (Bethyl lab), Phospho-Rpb1
CTD (Cell Signaling Technology), and H3K27ac (Abcam). Antibody-chromatin
complexes were captured with protein A and G Dynabeads (Thermo Fisher
Scientific) and washed with low salt wash buffer, high salt wash
buffer, and LiCl wash buffer. Chromatin-antibody immobilized on magnetic
beads were then subjected to tagmentation. Eluted DNA was purified using SPRI
Ampure XP beads (Beckman Coulter) and amplified for 8–12 cycles using
KAPA HiFi HotStart Ready mix (KAPA biosystems) and Nextera PCR primers
(Illumina). Libraries were purified using dual (0.65–0.25×) SPRI
Ampure XP beads (Beckman Coulter) and paired-end sequenced (100 bp) on the
Illumina HiSeq2500 platform.
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