1 × 107 cells were cross-linked with1% formaldehyde. The reaction was stopped using 0,125 M Glycine. After, the cells were lysed, in presence of protease inhibitors cocktail at 4 °C and the chromatin sonicated using Bioruptor (Diagenode) to obtain fragments of 200 bp. The immunoprecipitation was performed with the following antibodies: 5 μg of anti-HA (H6908, Sigma-Aldrich) (500 μg of protein), 5 μg of anti-H3K27me3 (#069-050, Diagenode) (200 µg of protein) and as control an irrelevant IgG (I5006, Sigma-Aldrich). The samples were incubated with Unblocked Protein A beads (C03020002, Diagenode), washed in columns (M1003S, MoBiTec) and incubated for 1 h at 37 °C. 200 mM NaCl solution was used to reverse cross-linking at 65 °C over night. After, the samples were treated with Proteinase K for 1 h at 55 °C, DNA was purified using columns of MiniElute PCR purification Kit (#28006, Quiagen, Valencia, CA) and eluted with DEPC water. The immunoprecipitated SDC-1 promoter fragments were quantified by real time PCR with the following primers: E-box 8-9-10, (forward) 5′-TTCCGCCCAGGAGAAAACAGAAAAG-3′, (reverse) 5′-CCTTTCCCTGCCTCTCTTACAGC-3′; E-box 5-6, (forward) 5′-GAGAGGTCGAGGCGATTCTCCC-3′, (reverse) 5′-TTTAAAAGTCACTCACGGCCAAG-3′; E-box 1, (forward) 5′-AACTTGTTCCTCTGCTGTGGATGGC-3′, (reverse) 5′-CACTCCCAACAGCAGTTATGAGCA-3′.
I5006
Manufactured by Merck Group
Sourced in United Kingdom
The I5006 is a laboratory equipment product. It is a device designed for use in scientific research and analysis. The core function of this product is to perform specific tasks or measurements within a laboratory setting. No further details on the intended use or features of this product can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Dynabeads protein a or g, by Thermo Fisher Scientific (2 mentions)
Hpa007415, by Merck Group (2 mentions)
Imprint rna immunoprecipitation kit, by Merck Group (2 mentions)
Ab9110, by Abcam (2 mentions)
Ab1791, by Abcam (2 mentions)
Anti ha, by Merck Group (1 mentions)
Bioruptor, by Diagenode (1 mentions)
Unblocked protein a beads, by Diagenode (1 mentions)
Anti h3k27me3, by Diagenode (1 mentions)
Anti histone h3, by Abcam (1 mentions)
Peroxidase antiperoxidase pap, by Merck Group (1 mentions)
Clone 42, by BD (1 mentions)
Gtx112346, by GeneTex (1 mentions)
Sc 166763, by Santa Cruz Biotechnology (1 mentions)
Dm2500, by Leica (1 mentions)
A21203, by Thermo Fisher Scientific (1 mentions)
Ab3672, by Abcam (1 mentions)
Ab50595, by Abcam (1 mentions)
Simplechip enzymatic chromatin ip kit, by Cell Signaling Technology (1 mentions)
Ab9110, by Merck Group (1 mentions)
23 protocols using i5006
1
ChIP-qPCR Analysis of H3K27me3
2
Evaluating Protein Expression in Yeast
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Saccharomyces cerevisiae (Sc) BY4741 strains containing C-terminally TAP-tagged genes (Open Biosystems, Germany) were tested for expression of the correctly tagged protein using the Peroxidase Anti-Peroxidase (PAP; Sigma, P1291, St. Louis, MO) antibody. To obtain the ΔPaf1 Set1-TAP strain, a ΔPaf1-KanMX6 cassette, amplified from the pFA6a-KanMX6 vector (Supplementary file 1 ), was introduced by homologous recombination into a Set1-TAP strain. A DNA fragment coding for Set1 residues 580 to 1080 and a C-terminal TAP tag was amplified from genomic DNA from a Set1-TAP strain (Supplementary file 1 ) and transformed into a ΔSet1 strain (Open Biosystems) by homologous recombination. Additional antibodies used were anti-Histone H3 (HRP; Abcam, ab21054, UK), anti-Histone H3 (tri methyl K4; Abcam, ab8580), anti-Histone H3 (tri methyl K36; Abcam, ab9050), anti-Histone H3 (tri methyl K79; Abcam, ab2621), IgG from rabbit serum (directed against the protein A content of the C-terminal TAP tag of proteins; Sigma, I5006), anti-rat IgG (HRP; Sigma, A9037) and anti-Ser2P (3E10; kindly provided by Dirk Eick [Chapman et al., 2007 (link)]).
3
Immunodetection of α-Synuclein Protein
4
Nuclear Protein Extraction and Immunoprecipitation
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Cells were rinsed twice with ice-cold HBSS and incubated with ice-cold hypotonic buffer (2 x 1min, 20mM HEPES, 20% Glycerol, 10mM NaCl, 1.5mM MgCl2, 0.2mM EDTA, 0.1% NP40, freshly supplemented with 1 mM DTT, and protease and phosphatase inhibitor cocktails). Nuclei were then harvested by scraping in hypertonic buffer (hypotonic buffer + 500mM NaCl, 400 µl/60 cm2) and disrupted by sonication in a water-bath sonicator. Nuclear lysates were cleared by centrifugation and quantified by Bradford. For immunoprecipitation, extracts were diluted to 140mM NaCl and incubated 4h at 4°C with magnetic beads (Dynabeads Protein A or G, Invitrogen) preloaded with specific primary antibodies. Immunocomplexes were then washed in binding buffer four times; finally, beads were resuspended in SDS sample buffer.
Antibodies used for immunoprecipitation: anti-BRD4 (E2A7X, CST); anti-YAP1 (13584-1-AP, Proteintech); anti-WWTR1 (HPA007415, Sigma); anti-FLAG (clone M2, A8592, Sigma); normal rabbit IgG (I5006, Sigma).
Antibodies used for immunoprecipitation: anti-BRD4 (E2A7X, CST); anti-YAP1 (13584-1-AP, Proteintech); anti-WWTR1 (HPA007415, Sigma); anti-FLAG (clone M2, A8592, Sigma); normal rabbit IgG (I5006, Sigma).
5
Immunoprecipitation of HMGCS1 Protein
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Whole-cell extracts of cells (1 × 105) were prepared as described above. To prepare the slurry of antibody-conjugated protein A-Sepharose, 1 μg of anti-HMGCS1 antibody (sc-166763, Santa Cruz) or rabbit IgG (I5006, Sigma-Aldrich), 50 μL of a 50% (vol/vol) slurry of protein A-Sepharose, and 450 μL of NETN buffer were incubated overnight at 4 °C under gentle rotation. Then whole-cell extracts (250 μg) and antibody-conjugated protein A-Sepharose were mixed overnight at 4 °C under rotary agitation. The immunoprecipitated proteins were eluted from the beads by boiling samples in sample buffer for Western blot analysis using anti-HMGCS1 (GTX112346, GeneTex) and anti-PERK (GTX81222, GeneTex) antibodies.
6
Subcellular Localization of Corin in HEK293 Cells
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HEK293 cells expressing corin were treated with CHX for 4 hr, fixed with 3% paraformaldehyde, permeabilized with 0.2% Triton X-100, and imminostained with antibodies against V5 (1:1000), PDI (1:200, Abcam, ab3672) or TGN46 (1:200, Abcam, ab50595) and Alexa Fluor-594 or 488-labeled secondary antibody (1:1000, Thermo Fisher, A-21203; A-11008). In controls, the primary antibody was replaced by mouse (Thermo Fisher, MA110419) or rabbit (Sigma, I5006) IgG. The stained cells were examined under a confocal microscope (Leica DM2500) and images were analyzed by ImageJ software.
7
RIP Assay for m6A RNA Binding
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RIP experiments were conducted in SW480 and HCT116 cells with the Imprint® RNA Immunoprecipitation Kit (RIP-12RXN, Sigma-Aldrich) following the manufacturer’s illustrations. Briefly, 4 μg specific antibodies against rabbit immunoglobulin G (I5006, Sigma-Aldrich), METTL14, YTHDF2 or m6A were incubated with cell lysates overnight at 4 °C with rotation, then 20 μl washed magnetic beads were added to each reaction and incubated at 4 °C for 2 h. After washed 6 times, interested RNAs in the immunoprecipitation complex were purified for further analysis by qRT-PCR. Relative enrichment of RNA was normalized to the input.
8
ChIP Assay of HA-tagged SOX6
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The ChIP assay was performed using Simple ChIP Enzymatic chromatin IP kit (Cell Signaling Technology, Danvers, Massachusetts, USA) as previously described [50 (link)]. Briefly, HeLa cells were transfected with plex-HA-SOX6 or plex-HA-SOX6ΔHMG plasmid. At 48 h post transfection, cells were treated with 1% methanol at room temperature for 10 min, and then added glycine to stop the reaction. After washing with cold PBS for three times, cells were lysed with SDS lysis buffer, and then the lysates were fragmented by ultrasound to get 300–500 bp DNA fragments in length. Finally, the lysates were incubated with rabbit anti-HA-tag-ChIP grade (Abcam, ab9110) or IgG from rabbit serum (Sigma-Aldrich, I5006).
9
Immunohistochemical Analysis of ANP32A
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Slides were deparaffinized in xylene and rehydrated in different concentrations of ethanol (100%, 95%, 90%, 80%, and 70%), and antigen retrieval performed using a citrate buffer. Slides were blocked (37°C, 2.5 h) with 10% fetal bovine serum and then incubated with anti-ANP32A antibody (2 μg/mL) for 12 hours at 4°C. IgG-purified normal rabbit serum (2 μg/mL) (I5006, Sigma, SF, USA) was used as a control. After washing in phosphate buffered saline, sections were incubated with polyperoxidase-anti-rabbit IgG for 30 min at 37°C. Signals were visualized by DAB oxidation and observed by Ti-s microscope (Nikon, Japan).
10
Immunofluorescence Analysis of Neutrophil Binding
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The control samples from the inhibition assay were stained to confirm by immunofluorescence microscopy the binding of the CD18a antibody onto the surface of the neutrophils. The permeabilization and the blocking for the staining were performed as above. Myeloperoxidase was detected using a polyclonal rabbit anti-human myeloperoxidase (A039829-2, 3.3 mg, 1:309; Agilent, Santa Clara, CA, USA). For the isotype controls, a rabbit IgG (from rabbit serum, I5006, 1.16 mg, 1:108.75; Sigma-Aldrich GmbH,) and a IgG1 Isotype Control from murine myeloma (M5284, 0.2 mg, 1:200; Sigma-Aldrich GmbH) were used. The first antibodies were incubated for 1 h at room temperature. As secondary antibodies, a goat anti-mouse IgG (H + L) DyLight 633 (#35512, 1:500; Invitrogen) and a goat anti-rabbit IgG (H + L) cross-adsorbed Alexa Fluor 488 (A11008, 1:500; Invitrogen) were used. The secondary antibodies were incubated for 1 h in the dark and at room temperature. The processing of the samples was carried out as explained above. The images were taken using a Leica TCS SP5 AOBS confocal inverted-base fluorescence microscope with an HCX PL APO 40× 0.75–1.25 oil immersion objective (see Figure A5 , Appendix A ).
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