Disuccinimidyl Glutarate

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.

9) 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.

10) 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.

11) 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.

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.

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.

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.

References:

[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.