Editing XIE/Enzyme:2025/Dec (section)

== 12.12 ==
*<big><span style="color:red;">'''S2 conjugated G5'''</span></big>
=== Incubation ===
'''Stock Solution''': 1 mM

# Measure G5 stock (3 mg/mL, 46 μM). Dilute 10 &mu;L of the stock to 100 &mu;L (4.6 μM); aliquot and store at -80&deg;C as the storage sample.
# Take one storage aliquot and add 10 &mu;L DBCO stock (10 mM). Divide evenly into two groups (ultrafiltration and non-ultrafiltration). For the non-ultrafiltration group, aliquot 16 &mu;L into a PCR tube (designated M1) and incubate on ice at 4&deg;C for 4 h.
# For the ultrafiltration group, transfer the sample into a 10 kDa centrifugal filter unit, dilute to 500 &mu;L with 1&times; PBS, and centrifuge at 10,000 &times; g, 4&deg;C for 30 min. Concentrate the sample to approximately 100 &mu;L, then spin at 8,000 &times; g for 2 min to collect. Aliquot 16 &mu;L into a PCR tube (designated M2).
# Add 1 &mu;L S1 (500 &mu;M) to both the ultrafiltered and non-ultrafiltered samples and incubate on ice at 4&deg;C for 4 h. Label the non-ultrafiltered sample N-U and the ultrafiltered sample U.
# Retain the −80&deg;C storage aliquot as control C.

=== Native PAGE ===
* Gel: 7.5% native gel.
* Sample preparation: Mix 16 &mu;L of each sample (C, M1, M2, N-U, U) with 4 &mu;L loading buffer and mix thoroughly.
* Electrophoresis: Run at 120 V for 100 min in 1&times; TBE.