2-Aminoethyl diphenylborinate

Biological Activity

2-Aminoethyl diphenylborinate (2-APB) is a cell-permeable inhibitor of IP3R, and regulate IP3-induced calcium release. 2-Aminoethyl diphenylborinate (2-APB) inhibits the SOC channel activity and activates TRP channel at higher concentrations.

DPBA staining solution preparation

Reagent Amount for 100 ml
2-Aminoethyl diphenylborinate (DPBA) 0.25 g
Triton X-100 20 μl
Milli-Q water  up to 100 ml
Notes:
a. Some crystals of DPBA can be retained and seen after mixing.
b. This staining solution can be stored at -20 °C for further uses.

Equipment

1. Tweezers
2. A plant growth chamber
3. Micro-tubes rotator
4. Pipette
5. Confocal microscopy system

Plant growth and DPBA staining procedure

1. Here, Arabidopsis thaliana is used. However, this protocol can be applied for other plants as well. Grow plants on MS medium supplemented with 2% sucrose and 1.2% phyto-agar for 5 days. The growth chamber conditions were 22 ± 1 °C, long-day (16 h light/8 h dark), and light intensity 100 μmol m^-2 s^-1.
Note: After seeding, remember to place the medium plates vertically in the growth chamber. Thereby, the roots grow on the surface of the medium and intact root tip samples can be obtained.
2. Add 1 ml of DPBA staining solution (Recipe 1) to each micro-tube.
3. Use a clean tweezers to carefully transfer the 5-day-old Arabidopsis seedlings to the micro-tube containing DPBA staining solution (5 seedlings/micro-tube).
4. Place the micro-tube in the rotator and rotate for 5 min at room temperature.
5. Stop the rotator, transfer the micro-tube to a rack and remove the DPBA staining solution.
Note: At this step, carefully use a pipette to remove the DPBA staining solution and try to not damage the plants, especially the root tips.
6. For washing, add 1.5 ml of distilled water to each micro-tube. Place the micro-tube in the rotator and rotate for 2 min at room temperature.
7. Stop the rotator, transfer the micro-tube to a rack and remove water.
8. Repeat Steps A6 and A7 two more times.
Note: After washing, can keep the plants in water and try to detect immediately. Do not leave the samples staying in water for longer than 30 min.
9. Transfer the seedlings to a microscope slide, cover it and detect the flavonols accumulation in the root tips by a confocal microscope.

Confocal microscopy

1. For DPBA-kaempferol, apply the emission spectrum (475-500 nm).
2. For DPBA-quercetin, apply the emission spectrum (585-619 nm).
3. Remember to take an additional bright field picture for control.

Chemical Information

Molecular Weight	225.09
Formula	C14H16BNO
CAS Number	524-95-8
Solubility (25°C)	DMSO ≥ 100 mg/mL
Storage	Powder          -20°C   3 years ;  4°C   2 years In solvent       -80°C   6 months ;  -20°C   1 month

References

[1] Mehmet Gazi Boyac, et al. World Neurosurg. Effects of 2-Aminoethyl Diphenylborinate, a Modulator of Transient Receptor Potential and Orai Channels in Subarachnoid Hemorrhage: An Experimental Study

[2] Kenji Takahashi, et al. Exp Cell Res. Molecular Mechanism of 2-APB-induced Ca²⁺ Influx in External Acidification in PC12

[3] Gergely Kovacs, et al. Cell Calcium. Inhibition of the Human Epithelial Calcium Channel TRPV6 by 2-aminoethoxydiphenyl Borate (2-APB)

[4] L Missiaen, et al. Cell Calcium. 2-Aminoethoxydiphenyl Borate Affects the Inositol 1,4,5-trisphosphate Receptor, the Intracellular Ca2+ Pump and the Non-Specific Ca2+ Leak From the Non-Mitochondrial Ca2+ Stores in Permeabilized A7r5 Cells

[5] Nguyen Hoai Nguyen. Bio Protoc. A Protocol for Flavonols, Kaempferol and Quercetin, Staining in Plant Root Tips