Histone extraction kit

Manufactured by Active Motif

The Histone Extraction Kit is a laboratory product designed to extract histones from biological samples. Histones are a group of proteins found in the nucleus of eukaryotic cells that play a crucial role in the organization and regulation of DNA. The kit provides the necessary reagents and protocols to efficiently isolate and purify histones from a variety of cell types and tissues.

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Lab products found in correlation

H3k27me3, by Cell Signaling Technology (2 mentions) Intercept pbs blocking buffer, by LI COR (2 mentions) Odyssey fc imaging system, by LI COR (2 mentions) Iblot 2 dry blotting system, by Thermo Fisher Scientific (2 mentions) Protease inhibitor cocktail, by Merck Group (2 mentions) Gapdh, by Santa Cruz Biotechnology (2 mentions) Bradford assay, by Bio-Rad (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Suz12, by Cell Signaling Technology (1 mentions) H3k27me2, by Merck Group (1 mentions) Premade polyacrylamide gels, by Thermo Fisher Scientific (1 mentions) H3k27me1, by Merck Group (1 mentions) H3r8me2s, by Epigentek (1 mentions) Rbbp4, by Fortis Life Sciences (1 mentions) Symmetric di methyl arginine, by Cell Signaling Technology (1 mentions) Prmt5, by Merck Group (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Il 6 elisa kit, by BioLegend (1 mentions) Image studio v5, by LI COR (1 mentions) Chameleon duo pre stained protein ladder, by LI COR (1 mentions)

9 protocols using histone extraction kit

Profiling Histone Modifications by Western Blot

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Protein samples were separated by electrophoresis on denaturing 4–12% premade polyacrylamide gels (Invitrogen) and blotted to PVDF membranes (Millipore). Membranes were blocked in TBST buffer plus 5% milk. Major antibodies: H3K27me3 (Cell Signaling, 9733), H3K27me2 (Millipore 07–452) and H3K27me1 (Millipore, 07–448), H3R8me2s (Epigentek A-3706–050), PRMT5 (Millipore 07–405), EZH2 (Cell Signaling 5246), Symmetric Di-methyl Arginine (Cell Signaling 13222), UTX (Cell Signaling 33510), EED (Millipore, 09–774), RBBP4 (Bethyl Laboratories A301–206A-T) and SUZ12 (Cell Signaling 1335947).
Histones were purified from mouse BM cells using Histone Extraction Kit (Active Motif, 40028) as per manufacturer's instructions. Successful purification of histones was confirmed by Coomassie staining before they were used for western blotting.

Liu F., Xu Y., Lu X., Hamard P.J., Karl D.L., Man N., Mookhtiar A.K., Martinez C., Lossos I.S., Sun J, & Nimer S.D. (2020). PRMT5-mediated histone arginine methylation antagonizes transcriptional repression by polycomb complex PRC2. Nucleic Acids Research, 48(6), 2956-2968.

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Quantifying Immune Modulators and Histone Modifications

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The supernatants of cultured and ablated KPC and S2013 were analyzed using murine and human MIF ELISA kits (R&D Systems). Supernatant from in vivo trained ex vivo stimulated CD11b+ cells were analyzed using murine TNF-α and interleukin (IL)-6 ELISA kits (BioLegend). To detect histone modification, histones were extracted from CD11b+ cells, which were positively selected from PBS, β-glucan, IRE, and β-glucan plus IRE treated KPC tumors using the Histone Extraction Kit (Active Motif, Catalong # 40028). Histone modifications were quantified using EpiQuik Total Histone H3, Acetyl histone H3K27, Tri-Methyl histone H3K4, and Tri-methyl histone H3K27 Quantification Kits (Colorimetric). Supernatants from in vitro trained human CD14+ cells were analyzed using human TNF-α ELISA kits (BioLegend). All assays were performed using the provided manufacturer instructions and all conditions were performed in duplicates.

Woeste M.R., Shrestha R., Geller A.E., Li S., Montoya-Durango D., Ding C., Hu X., Li H., Puckett A., Mitchell R.A., Hayat T., Tan M., Li Y., McMasters K.M., Martin R.C, & Yan J. (2023). Irreversible electroporation augments β-glucan induced trained innate immunity for the treatment of pancreatic ductal adenocarcinoma. Journal for Immunotherapy of Cancer, 11(4), e006221.

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Comprehensive Protein Extraction and Analysis

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Total protein extraction was performed using lysis buffer (Tris-HCl 50 mmol/L pH 8, NaCl 150 mmol/L, Triton-X-100 1%, NaF 100 mmol/L, EDTA 1 mmol/L, MgCl₂ 1 mmol/L, and glycerol 10%) containing a protease inhibitor cocktail (Sigma-Aldrich). Equal amounts of total protein were subjected to Bis-Tris gel electrophoresis (Nupage) and then transferred to nitrocellulose membranes using the iBlot2 Dry Blotting System (Thermo Fisher). The membranes were blocked with Intercept PBS Blocking Buffer (Li-Cor) and incubated with primary antibodies overnight at 4°C (Supplementary Table S9). After washing, membranes were incubated with Li-Cor Fluorescent IRDye secondary antibodies at room temperature (800 CW and 680 CW dyes) for 1 hour. Detection was performed with the Li-Cor Odyssey FC Imaging System. Histone extraction was performed using the Histone Extraction Kit (Active Motif). Cells were lysed according to the one-step extraction protocol provided by the manufacturer, and histone lysates then underwent Bis-Tris gel electrophoresis, transfer, and detection as described above.

McNicholas M., De Cola A., Bashardanesh Z., Foss A., Lloyd C.B., Hébert S., Faury D., Andrade A.F., Jabado N., Kleinman C.L, & Pathania M. (2023). A Compendium of Syngeneic, Transplantable Pediatric High-Grade Glioma Models Reveals Subtype-Specific Therapeutic Vulnerabilities. Cancer Discovery, 13(7), 1592-1615.

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Western Blot and Histone Extraction

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Total protein extraction was performed using lysis buffer (Tris-HCl 50 mM pH 8, NaCl 150 mM, Triton X-100 1%, NaF 100 mM, EDTA 1 mM, MgCl₂ 1 mM, Glycerol 10%) containing a protease inhibitor cocktail (Sigma-Aldrich). Equal amounts of total protein were subjected to Bis-Tris gel electrophoresis (Nupage) and then transferred to nitrocellulose membranes using iBlot2 Dry Blotting System (Thermo Fisher). The membranes were blocked with Intercept PBS Blocking Buffer (Li-Cor) and incubated with primary antibodies overnight at 4°C (Supplementary Table 9). After washing, membranes were incubated with Li-Cor Fluorescent IRDye secondary antibodies at RT (800 CW and 680 CW dyes) for 1 hour. Detection was performed with Li-Cor Odyssey FC Imaging System. Histone extraction was performed using the Histone Extraction Kit (Active Motif). Cells were lysed according to the One-step Extraction protocol provided by the manufacturer and histone lysates then underwent Bis-Tris gel electrophoresis, transfer and detection as described above.

Lane B., Challen K., Harris H.J, & Harris R. (2001). Existence and quality of written antenatal screening policies in the United Kingdom: postal survey. BMJ : British Medical Journal, 322(7277), 22-23.

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Histone Extraction and Western Blot Analysis

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Confluent HFFs cultured in a six-well plate were treated with indicated concentrations of GSK-J4 or UNC1999 in 10% FBS DMEM for 4 days, with a media change including fresh inhibitor on day 2. Untreated cells were cultured in parallel. Histones were isolated from inhibitor-treated or untreated cells using the histone extraction kit (Active Motif, 40028), and western blots were performed. Histone extracts were combined with Li-cor 4X Protein Loading Buffer (928-40004) and resolved on Bio-Rad Mini-PROTEAN TGX 4-20% gel (4561094) in Boston BioProducts Tris-Glycine-SDS Running Buffer (BP-150), and transferred onto an Immobilon-FL PVDF membrane (IPFL00010) using Boston BioProducts Transfer Buffer (BP-190) made to 20% vol/vol methanol. Membranes were blocked in Odyssey Blocking Buffer (OBB, 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 2% Fish gelatin, 1% ovalbumin) for a minimum of 1 hour at room temperature, washed with TBS-T (Research Products International T60075-4000.0 with 0.1% Tween 20, and incubated with primary antibody (diluted in OBB with 0.2% Tween 20) overnight at 4°C. Li-cor secondary antibodies (925-32211, 925-68070) were diluted in OBB (with 0.2% Tween 20, 0.02% SDS) and blots imaged on a Li-Cor Odyssey CLX-1374. Band intensity quantification was performed using Li-Cor Image Studio v5.2. Li-Cor Chameleon Duo Pre-Stained Protein Ladder (928-60000) was used.

Francois A.K., Rohani A., Loftus M., Dochnal S., Hrit J., McFarlane S., Whitford A., Lewis A., Krakowiak P., Boutell C., Rothbart S.B., Kashatus D, & Cliffe A.R. (2024). Single-genome analysis reveals a heterogeneous association of the herpes simplex virus genome with H3K27me2 and the reader PHF20L1 following infection of human fibroblasts. mBio, 15(4), e03278-23.

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Inducible Fusion Protein Overexpression and Histone Analysis

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Fusion protein overexpression was induced by treatment with doxycycline (dox) (Sigma Aldrich) at a concentration of 1 µg/mL for the specified amount of time. Pellets were harvested for western blot analysis and washed with PBS (Corning, 21-040-CV). Cells were subsequently lysed on ice using RIPA buffer (Boston BioProducts, BP-115-250) supplemented with fresh HALT Protease Inhibitor with EDTA (Thermo Fisher Scientific, 78429), and the lysates were clarified through centrifugation. For histone immunoblotting, histones were extracted according to the recommended manufacturer’s one-step protocol using the Histone Extraction Kit (Active Motif, 40028). The protein concentration of the lysates was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific, 23225). Immunoblotting was performed according to standard procedures. To assess histone loading, the blot was stripped with Restore Western Blot Stripping Buffer (Thermo Fisher Scientific, 20159) for 30 min prior to re-probing for Histone H3. The primary antibodies used for immunoblotting are as follows: GAPDH (1:5000, Santa Cruz Biotechnology sc-477724); monoclonal anti-FLAG M2 (1:1000 and 1:5000, Sigma-Aldrich F1804); Tri-Methyl-Histone H3 Lys27 (1:10000, Cell Signaling Technology C36B11); Histone H3 (1:10000, Cell Signaling Technology 9715 S); and EZH2 (1:4000, Cell Signaling Technology 5246).

Siegenfeld A.P., Roseman S.A., Roh H., Lue N.Z., Wagen C.C., Zhou E., Johnstone S.E., Aryee M.J, & Liau B.B. (2022). Polycomb-lamina antagonism partitions heterochromatin at the nuclear periphery. Nature Communications, 13, 4199.

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Histone Extraction and Immunoblotting

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For whole cell extracts, cells were lysed on ice using radioimmunoprecipitation assay (RIPA) buffer (Boston BioProducts) supplemented with fresh HALT^™ protease inhibitor cocktail (Thermo Fisher Scientific). The lysate was clarified by centrifugation at 18,000 x g for 15 min. For isolation of core histones, lysates were prepared using the Histone Extraction Kit (Active Motif). Protein concentration of the lysates was measured using Bradford Assay (Bio-rad). Immunoblotting was performed according to standard procedures. The primary antibodies used are as follows: EZH2 (Cell Signaling Technology, #5246; 1:5,000 dilution); H3K27me1 (Active Motif, cat no. 61015; 1:5,000 dilution); H3K27me2 (Cell Signaling Technology, #9728; 1:5,000 dilution); H3K27me3 (Cell Signaling Technology, #9733; 1:5,000 dilution); H3K36me3 (Cell Signaling Technology, #9763); Histone 3 (Active Motif, cat no. 39763; 1:10,000 dilution); GAPDH (Santa Cruz Biotechnology, sc-477724; 1:10,000 dilution).

Takebayashi Y., Pourquier P., Yoshida A., Kohlhagen G, & Pommier Y. (1999). Poisoning of human DNA topoisomerase I by ecteinascidin 743, an anticancer drug that selectively alkylates DNA in the minor groove. Proceedings of the National Academy of Sciences of the United States of America, 96(13), 7196-7201.

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Chromatin Profiling of GBM Samples

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The snap-frozen brain tissue samples were homogenized in 1 ml Buffer A (10 mM Tris-HCl pH = 8, 250 mM Sucrose, 10 mM MgCl2, 1 mM EGTA) with 1X protease inhibitor to extract cytoplasmic proteins. The resulting pellets were further homogenized in 1 ml RIPA buffer containing 1X protease inhibitor to isolate nuclear fraction. The total protein concentrations in cytoplasmic and nuclear fractions were quantified by Bradford assay (Bio-Rad). Bulk chromatin extraction was performed with histone extraction kit from Active Motif. For western blot, equal amounts of protein from normal or GBM specimens were denatured in final 1X SDS stop buffer and subjected to SDS-PAGE for western blot analysis with antibodies against H3K4me3 (Millipore #07-473; 1:1000), H3, H4, MLL1, MLL2, MLL3, and γ-tubulin (Sigma #T5326; 1:1000). Subsequently, HRP-conjugated secondary IgG (Cell Signalling; 1:2000) and enhanced chemiluminescence kit (ECL plus; GE) were used for detection. Of note, blotting membranes were cut into strips to accommodate the molecular weights of MLL, H3K4me3, and γ-tubulin for antibody incubation separately. Anti-H3K4me3 was stripped after ECL detection and the blots were subsequently incubated with unmodified H3 and H4 antibodies (internal controls).

Sandstrom R.S., Foret M.R., Grow D.A., Haugen E., Rhodes C.T., Cardona A.E., Phelix C.F., Wang Y., Berger M.S, & Lin C.H. (2014). Epigenetic Regulation by Chromatin Activation Mark H3K4me3 in Primate Progenitor Cells within Adult Neurogenic Niche. Scientific Reports, 4, 5371.

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Histone Modification Analysis in Keratinocytes

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Histones were extracted from KCs and the epidermis using a histone extraction kit (No. 40028, Active Motif) according to the manufacturer's instructions. Proteins were loaded on 10% SDS-polyacrylamide gel electrophoresis (PAGE) gels, transferred to polyvinylidene fluoride membranes (Millipore, USA), and visualized by Western blotting using the following specific antibodies: anti-H3K9me3 (1:1,000; #13969, CST), anti-H3K4me3 (1:1,000; #9751, CST), anti-H3K27me3 (1:1,000; #9733, CST), anti-H3K36me3 (1:1,000; #9763, CST), and anti-histone H3 (1:1,000; #4499, CST). The blots were imaged using a gel image analysis system (Bio-Rad, Hercules, CA, USA).

Chen C., Yi X., Liu P., Li J., Yan B., Zhang D., Zhu L., Yu P., Li L., Zhang J., Kuang Y., Zhao S., Zhu W., Peng C, & Chen X. (2023). CD147 Facilitates the Pathogenesis of Psoriasis through Glycolysis and H3K9me3 Modification in Keratinocytes. Research, 6, 0167.

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