Disuccinimidyl Glutarate is an insoluble bisfunctional N-hydroxysuccinimide (NHS ester) crosslinking agent that is commonly used to pair radiolabelled ligands to cell surface receptors[1].
Combining Disuccinimidyl Glutarate and formaldehyde crosslinking is essential to detect enrichment of TET2 on chromatin. Inclusion of the protein protein crosslinker Disuccinimidyl Glutarate as well as titration of antibody and input chromatin resulted in improved signal-to-noise ratio[1]. When treated embryos with increasing concentrations of DSG (1.0 -5 mM) in the presence of an equal volume of heptane. After vigorously shaking the DSG-embryo suspension for 1 h, formaldehyde was added so that the final concentration in the water phase would be 4%. After a 15 min incubation with formaldehyde, the embryos were processed including the nuclear formaldehyde cross-linking step. The DSG-formaldehyde fixation is much more effective in capturing Elba association with Fab-7 in vivo than formaldehyde alone. The Elba1 antibody ChIP for both the 5.0 mM and 2.5 mM Disuccinimidyl Glutarate -treated embryos showed an appreciable enrichment (6-7 fold) of Fab-7 compared with the pre-immune control. The pull down of Fab-7 sequences appears to be specific as the 2 control loci, twe and Sxl, showed only a 1-1.5 fold enrichment. While there wasn t much difference between the 5.0 mM and the 2.5 mM DGS fixation, having a sufficiently high concentration of this cross-linking reagent does seem to be important, as there was only a limited enrichment with 1.0 mM Disuccinimidyl Glutarate cross-linking[2].
参考文献:
[1]. Rasmussen KD, Helin K. ChIP-Sequencing of TET Proteins. Methods Mol Biol. 2021;2272:251-262. doi: 10.1007/978-1-0716-1294-1_15. PMID: 34009619.
[2]. Aoki T, Wolle D, et,al. Bi-functional cross-linking reagents efficiently capture protein-DNA complexes in Drosophila embryos. Fly (Austin). 2014;8(1):43-51. doi: 10.4161/fly.26805. Epub 2013 Dec 13. PMID: 24135698; PMCID: PMC3974894.
Disuccinimidyl Glutarate 是一种不溶性双功能 N-羟基琥珀酰亚胺（NHS 酯）交联剂，通常用于将放射性标记的配体与细胞表面受体配对[1]。
结合戊二酸二琥珀酰亚胺酯和甲醛交联对于检测 TET2 在染色质上的富集至关重要。加入蛋白质交联剂戊二酸二琥珀酰亚胺酯以及滴定抗体和输入染色质可提高信噪比[1]。在等量庚烷存在的情况下，用增加浓度的 DSG (1.0 -5 mM) 处理胚胎。剧烈摇动 DSG-胚胎悬浮液 1 小时后，加入甲醛，使水相中的最终浓度为 4%。用甲醛孵育 15 分钟后，对胚胎进行处理，包括核甲醛交联步骤。 DSG-甲醛固定在体内捕获 Elba 与 Fab-7 的结合比单独使用甲醛更有效。与免疫前对照相比，5.0 mM 和 2.5 mM 戊二酸二琥珀酰亚胺酯处理的胚胎的 Elba1 抗体 ChIP 显示 Fab-7 明显富集（6-7 倍）。 Fab-7 序列的下拉似乎是特定的，因为 2 个控制基因座 twe 和 Sxl 仅显示 1-1.5 倍的富集。虽然 5.0 mM 和 2.5 mM DGS 固定之间没有太大差异，但具有足够高浓度的这种交联剂似乎很重要，因为 1.0 mM 戊二酸二琥珀酰亚胺酯交联的富集度有限< sup>[2].
| Cross-linking of embryos for CHIP-Seq [1]: | |
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Preparation Method |
1)After dechorionation, the embryos were weighed and transferred to a 50 ml conical tube. If there was more than 2 g of embryos, we split the sample into multiple tubes. 2) The bi-functional NHS-esters were dissolved in DMSO (Dimethyl sulfoxide). In the case of Disuccinimidyl Glutarate（DSG）, we dissolved 20 mg of DSG powder in 108 ul of DMSO to give -120 ul of a 500mM DSG stock solution. In the case of DSP, we dissolved 20 mg of powder in 85 ul of DMSO to give -99 ul of 500 mM DSP stock solution. 3) The fixation mix was prepared by adding the NHS-ester/DMSO solution to phosphate-buffered saline (PBS, 150 mM sodium chloride (NaCl) / 10 mM sodium phosphate (pH 7.6)). The optimal concentration of NHS-esters may differ between the target proteins. The maximum concentrations of Disuccinimidyl Glutarate and DSP in aqueous solution are 5 mM and 2 mM, respectively. For less than 0.5 g of embryo, prepare 5 ml of solution. Increase the volume by 5 ml for every 0.5 g of additional amount of embryo. Because the NHS-esters are unstable in aqueous solutions, this fixation mix should be prepared immediately before use. 4) Pour the fixation mix into the conical tube containing the dechorionated embryos. Add an equal volume of heptane. 5) Shake the tube vigorously for 1 h. 6) Add 550 μl of 37% formaldehyde per 5 ml of aqueous phase so that the final concentration of formaldehyde 4%. Shake vigorously for additional 15 min. 7) Centrifuge the tube in swinging bucket rotor at 500 g for one minute to pellet the fixed embryo. Remove the heptane phase by pipetting. 8) Add an equal volume of the stop solution (PBS with 125 mM Glycine and 0.1% Triton X-100) to the aqueous phase. Mix well, let stand for 2 min and then pellet the fixed embryos by centrifuging a 500 g for 1 min. 8) Remove the supernatant by pipetting. Suspend the embryos in 25 ml of stop solution and after a 2 min incubation collect the embryos by centrifugation. Spin again in the same condition. 9) Remove the supernatant, and wash the embryos by suspending in 25 ml of PBST (PBS + 0.05% Triton X-100) and spin again in the same condition. Repeat this step 2 times. 10) The fixed embryos can either be immediately processed for ChIP or transferred to a 1.5ml tube, frozen in liquid nitrogen and then stored at -80 ℃ until use. |
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Applications |
The Disuccinimidyl Glutarate -formaldehyde fixation is much more effective in capturing Elba association with Fab-7 in vivo than formaldehyde alone. The Elba1 antibody ChIP for both the 5.0 mM and 2.5 mM DSG-treated embryos showed an appreciable enrichment (6-7 fold) of Fab-7 compared with the pre-immune control. |
| Cell Crosslinking [2]: | |
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Preparation Method |
1. Remove a new vial of DSG (disuccinimidyl glutarate) from 4 ℃ storage. When it is equilibrated to room temperature, resuspend the desiccated powder in DMSO to obtain a stock solution of 0.25 M Disuccinimidyl Glutarate. Immediately before adding to the cells, prepare the final crosslinking solution of 2 mM Disuccinimidyl Glutarate in ice-cold PBS (e.g., 80 μL of 0.25 M DSG in 10 mL of PBS). Discard the unused 0.25 M Disuccinimidyl Glutarate stock solution as reconstituted Disuccinimidyl Glutarate will tend to hydrolyze and become inactive during storage. 2. Resuspend cell pellet from each sample in 10 mL of freshly prepared, ice-cold 2 mM DSG crosslinking solution. Place tubes on a roller mixer and allow crosslinking to proceed for 30 min while the solution is equilibrating to room temperature. 3. After the initial 30 min has passed, add formaldehyde to a final concentration of 1% (e.g., 270μl of a 37% stock solution in 10 mL). Incubate on a roller mixer at room temperature for an additional 10 min. 4. Immediately after 10 min has passed, quench crosslinking reaction by adding 2 M glycine to a final concentration of 0.125 M (e.g., 625 μL in 10 mL). Incubate on a roller mixer at room temperature for an additional 5 min. 5. Spin cells (300 g/5 min/4 ℃) and wash twice with ice-cold PBS. Discard solution containing formaldehyde in the appropriate waste container. Proceed immediately to lysis and sonication. |
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Applications |
Combining Disuccinimidyl Glutarate and formaldehyde crosslinking is essential to detect enrichment of TET2 on chromatin. Inclusion of the protein protein crosslinker DSG as well as titration of antibody and input chromatin resulted in improved signal-to-noise ratio. |
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参考文献: [1]. Aoki T, Wolle D, Preger-Ben Noon E, Dai Q, Lai EC, Schedl P. Bi-functional cross-linking reagents efficiently capture protein-DNA complexes in Drosophila embryos. Fly (Austin). 2014;8(1):43-51. doi: 10.4161/fly.26805. Epub 2013 Dec 13. PMID: 24135698; PMCID: PMC3974894. [2]. Rasmussen KD, Helin K. ChIP-Sequencing of TET Proteins. Methods Mol Biol. 2021;2272:251-262. doi: 10.1007/978-1-0716-1294-1_15. PMID: 34009619. |
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