Western blots to visualize Hda1 and H2Bub were performed using whole-cell extracts prepared using SUTEB buffer as described (Van Oss et al. 2016 (link)). H3 methylation was examined using whole-cell extracts prepared using a TCA extraction method (Van Oss et al. 2016 (link)). Proteins were run on 15% SDS-polyacrylamide gels. The following antibodies were used: α-Hda1 (Santa Cruz sc-393814; 1:200), α-H2B (Active Motif #39237; 1:3000), α-H2Bub (Cell Signaling #5546; 1:1000), α-G6PDH (Sigma #A9521; 1:20000), α-H3K4Me2 (Millipore #07-030; 1:2000), α-H3K4Me3 (Active Motif #39159; 1:2000), α-H3K79Me2/3 (Abcam #ab2621; 1:1000; note that this antibody recognizes both di- and trimethylated H3K79) and α-H3 antibody (Tomson et al. 2011 (link)). Blots were developed using Thermo Scientific West Pico Plus (34580) and images were collected on a BioRad ChemiDoc™ XRS+.
α h3k4me3
Manufactured by Active Motif
α-H3K4me3 is an antibody that specifically recognizes the trimethylation of lysine 4 on histone H3. This epigenetic mark is associated with active transcription and is commonly used to identify regions of active gene expression.
Automatically generated - may contain errors
Lab products found in correlation
α g6pdh, by Merck Group (3 mentions)
α h3k4me2, by Merck Group (3 mentions)
α flag, by Merck Group (3 mentions)
α h2b, by Active Motif (2 mentions)
α h2bub, by Cell Signaling Technology (2 mentions)
α h3k79me2 3, by Abcam (2 mentions)
Chemidoc xr, by Bio-Rad (2 mentions)
α h3k4me1, by Active Motif (2 mentions)
Glutathione agarose column, by Merck Group (1 mentions)
α ubiquitin, by Santa Cruz Biotechnology (1 mentions)
Ab46765, by Abcam (1 mentions)
Mca1360, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Cell Signaling Technology (1 mentions)
α pmad, by Abcam (1 mentions)
α h3k9me3, by Active Motif (1 mentions)
αh3k27me3, by Active Motif (1 mentions)
Lsm 700 confocal microscope, by Zeiss (1 mentions)
Mouse α gfp antibody, by Santa Cruz Biotechnology (1 mentions)
Goat α rabbit, by Bio-Rad (1 mentions)
Secondary goat α mouse, by Bio-Rad (1 mentions)
8 protocols using α h3k4me3
1
Visualizing Histone Modifications by Western Blot
2
Antibody Production and Characterization
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Rabbit and guinea pig anti-Cmi/Lpt polyclonal sera (Pocono Rabbit Farm) were raised against amino acids residues 647–1069 fused to GST. The induced protein was purified on a glutathione agarose column (Sigma) and used for antibody production. Rabbit αSnr1 antibody was prepared as described (46 (link)). Mouse monoclonal antibody αRNA Polymerase II 8W (MMS-126R) was from Covance. Rabbit αH3 antibody (ab1791), αIgG (ab27478) and αHA (ab9110) were from Abcam. Rabbit αH3K27ac (39 133), αH3K27me3 (39 155), αH3K4me1 (39297) and αH3K4me3 (39 915) were from Active Motif. Lamin antibody (ADL40) was obtained from the Developmental Studies Hybridoma bank at the University of Iowa. Rabbit αTrr and rabbit αCBP antibodies were from A. Mazo (47 (link)). Rabbit αUtx antibody was a gift from A. Shilatifard (48 (link),49 (link)). Guinea pig αCBP antibody was a gift from P. Harte (50 (link)). Rabbit αGrh (grainyhead) antibody was a gift from M. Harrison (51 (link)) and guinea-pig αGrh antibody was a gift from W. McGinnis.
3
Antibody Characterization in Biochemical Assays
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Antibodies used are as follows: α-HA (Santa Cruz, sc-7392), α-ubiquitin (Santa Cruz, sc-8017), α-FLAG (Sigma, #F1804), α-G6PDH (Sigma, #A9521), α-H3 (Active Motif, #39163), α-H3K4me3 (Active Motif, #39159), α-RNA Polymerase II (Active Motif, #39097), α-Rpt2 (Enzo Life Sciences, #BML-PW8260), and α-20S (Enzo Life Sciences, #BML-PW8195). The α-Rpt6 antibody was described previously25 (link).
4
Western Blot Analysis of H3K4 Methylation
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To examine H3K4 methylation or epitope-tagged H3, cell extracts were prepared following the bead-beat procedure as described66 (link). A twofold serial dilution of extracts were resolved in 15% SDS–PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane. The following antibodies were used in western blotting (dilutions used, source and catalogue numbers are indicated within parentheses): α-H3K4me1 (1:1,000, Active Motif, 39,297), α-H3K4me2 (1:10,000, Millipore, 07–030), α-H3K4me3 (1:7,500, Active Motif, 39,159), α-V5 (1:50,000, Bio-Rad, MCA1360) and α-Flag (1:5,000, Sigma, F3165). The histone H3 loading was monitored using α-H3 (1:2,000, Abcam, ab46765). Full-length images of the blots are shown in Supplementary Figs 20–22 .
5
Immunostaining of Drosophila Imaginal Discs
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Imaginal discs were dissected and fixed in 4% paraformaldehyde for 20 min, washed and permeabilized in phosphate-buffered saline with 0.1% Triton X-100, and blocked in 10% normal goat serum. Primary antibodies used were α-Ci [1:25; Developmental Studies Hybridoma Bank (DSHB)], α-Ubx (1:10; DSHB), α-En (1:25; DSHB), α-β-galactosidase (1:500; Promega), α-V5 (1:500; Sigma-Aldrich), α-Wg (1:100; DSHB), α-Cut (1:100), α-Ptc (1:50; DSHB), α-pMAD (1:500; Abcam), α-Smo (1:10; DSHB), α-MMP1 (1:100; a combination of 14A3D2, 3A6B4, and 5H7B11; DSHB), α-Tara (1:700; K. Koh), α-H3K27me3 (1:500; Active Motif), α-H3K9me3 (1:500; Active Motif), α-H3K4me1 (1:500; Active Motif), α-H3K4me3 (1:500; Active Motif), α-H4ac (1:500; Active Motif), and α-Sd (1:500; K. Guss). Secondary antibodies were from Cell Signaling Technology and used at 1:400. Nuclei were stained with 4′,6-diamidino-2-phenylindole (1:1000; Cell Signaling Technology). Samples were imaged on a Zeiss LSM 700 confocal microscope.
6
Immunoblotting and Nuclear Fractionation
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For GFP-tagged KdmB, mouse α-GFP antibody (Cat# SC-9996, SantaCruz) was used at 1:1,000 dilution in blocking solution (TBST with 5% milk). Secondary goat α-mouse (Cat# 170–6516, Biorad) was used at 1:2,000 dilution in blocking solution. A rabbit polyclonal antibody against a subunit of the SCF complex, SkpA (SconC) was used (1:2,000 dilution in TBST with 5% milk) as a loading control with the Goat α-Rabbit (Bio-Rad, Cat# ab190479; 1:2,000) secondary antibody. For nuclear isolation, approximately 2x106 spores were inoculated into GMM with required supplements for 24 h at 30 °C submerged culture. Buffers and extraction protocol were performed similarly as described previously (Soukup and Keller, 2013 ). Antibodies (1:2,000 dilution in blocking solution) used for histone PTMs were as following; α-H3 (Abcam; AB1791), α-H3K4me3 (Active Motif; 39159), α-H3K9me3 (Abcam; ab8898), α-H3K36me3 (Abcam; ab9050), α-H3K9ac (Merck; 07–352), α-H3K14ac (Merck; 07–353).
7
Chromatin Profiling using CUT&RUN and Immunoblotting
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Antibodies used for CUT&RUN are αH2A (Abcam, 15653), αFLAG (Sigma, F1804), αmouse rb IgG (Jackson ImmunoResearch, 315-005-003). Primary antibodies used for immunoblotting include: αγH2Ax (Milipore, ab26350), αFLAG (Sigma, F7425), αH2B (CST, D2H6), αH4 (CST, D2X4V), αH3 (Immunoway, YM3038), αNAP1L1 (Abcam, ab33076), αNAP1L2 (Abnova, H00004674-D01), αSPT16 (Santa Cruz, sc-377028), αSSRP1 (Santa Cruz, sc-74536), αHSC70 (Santa Cruz, sc-7298), αC23 (Santa Cruz, sc-55486), αH2BK120ub (CST, D11), αH2AK119ub (CST, D11), αH3K4me3 (Active Motif, 61379), αH3K36me3 (Immunoway), αH3K36me2 (CST, 29015), αH3K9me3 (Abcam, ab8898), αH3K27me3 (CST, 9733), αH4K16ac (Immunoway, YK0014), αH4K20me3 (Active Motif, 39671), αH3K27ac (Active Motif, 39685), αH3K79me3 (Immunoway, YM3091), αH3K9ac (Immunoway, YK0006), αHA (Santa Cruz, SC7392), αβ-Actin (Immunoway, YM3028). αFLAG M2 Affinity Gel (Sigma), αH2A (Abcam, 15653), and αRpb1 (CST, 14958) were used for ChIP.
8
Visualizing Histone Modifications by Western Blot
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Western blots to visualize H2Bub were performed using whole-cell extracts prepared using SUTEB buffer as described (Van Oss et al. 2016) . H3 methylation was examined using whole-cell extracts prepared using a TCA extraction method (Van Oss et al. 2016) . Proteins were run on 15% SDS-polyacrylamide gels. The following antibodies were used: α-H2B (Active Motif #39237; 1:3000), α-H2Bub (Cell Signaling #5546; 1:1000), α-G6PDH (Sigma #A9521; 1:20000), α-H3K4Me2 (Millipore #07-030; 1:2000), α-H3K4Me3 (Active Motif #39159; 1:2000), α-H3K79Me2/3 (Abcam #ab2621; 1:1000; note that this antibody recognizes both di-and trimethylated H3K79), and α-H3 antibody (Tomson et al. 2011) . Blots were developed using Thermo Scientific West Pico Plus (34580) and images were collected on a BioRad ChemiDoc™ XRS+.
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