Editing XIE/Enzyme:2025/Dec (section)

== 12.15 ==
*'''<big><span style="color:red;">S2 conjugated enzyme</span></big>'''
=== Sample preparation ===
* Bis-sulfone-PEG4-DBCO:dilute 2 µL DBCO in PBS (pH 7.5) to a final volume of 20 µL (1 mM). Incubate at 37 °C for 1 h to convert bis-sulfone to mono-sulfone (optimal pH ≈ 7.4).
*Enzyme:
#Prepare stocks of G5, Al, SS, RA, and RH (120 µL at 10 µM each). Dilute each stock to 240 µL with PBS (pH 7.0) to obtain 5 µM working solutions. (the optimal pH for the enzyme–DBCO reaction is 6.5–7.0.)
#Take 40 µL of each diluted G5, Al, SS, RA, and RH to use as parallel control samples.

=== Reaction ===
*Add 4 µL of the aqueous DBCO solution to each of G5, Al, SS, RA, and RH and incubate for 4 h. (Enzyme:DBCO ratio = 1:4.)
*Split each sample evenly into two aliquots: designate one as the M control, and subject the other to ultrafiltration and washing to remove excess reagents and ensure the final composition is unchanged. Incubate the samples on ice for 4 h.

=== SDS page ===
*Gel:10% concentration
*Running Buffer:Tris:3.03g、Glycine:14.4g、SDS:1g、Water:1 L.
*Stain:Coomassie blue 37℃ Sway
*Sample:Marker, C-G5, G5, C-Al, M-Al, Al, C-SS, M-SS, SS, C-RA, M-RA, RA, C-RH, M-RH, RH.

=== Result and Problem ===
*When DBCO (dissolved in DMF) is diluted into PBS, precipitation occurs.