Protocol: Difference between revisions

Revision as of 12:01, 7 November 2025

Proteins containing 6x Histidine at the C terminus reacted, with the chemical DBCO-PEG4-Bis-sulfone(DPBS) for 4 h at room temperature. The bis-sulfone group reacts with the Histidines at the C terminus of the protein, and then an azide modified oligonucleotide is conjugated to the protein by click chemistry between the azide- and the DBCO-group.

conjugation: DNA Origami = 20:1, and incubate in a PCR machine with a temperature ramp starting from 1 h at 37 ℃ follow by 14 h at 22 ℃, and immediately after incubation the nanopatterns are stored at 4 ℃.

Sample
Reagent	Sequence
DBCO-PEG4-Bis-sulfone
azide modified oligonucleotide	CTCTCCTTCTTCCCTTTCTTT
azide modified oligonucleotide	TTCGACAGCATGAACATCAGC

取 1.4 mg GDH 溶解于 2 mL 1×PBS 中。

样品	原样品浓度	终样品浓度	加入量	总体积
GDH	0.7 mg/mL	0.175 mg/mL	25 μL	100 μL

样品	初始浓度	取样量	最终浓度	最终体积
RuCHMO	28.2 μM	100 μL	5 μM	564 μl

取 DBCO 2 mg 溶解DMF 20 ul 中

样品	原样品浓度	终样品浓度	加入量	总体积
DBCO	100 mM	20 mM	2 μL	20 μL

样品	最终浓度	分子量	总摩尔量	Eq DBCO	DBCO 摩尔量	DBCO 加入量	总体积
GDH	5 mM	35 kDa	0.5 nMol	20 Eq	10 nMol	1 μL	100 μL

Name	序列	bp	修饰	Nmol/管	buffer (μL)	浓度 (mM)
S1	CTCTCCTTCTTCCCTTTCTTT	21	5'Azide	123.53	123.53	1
S2	TTCGACAGCATGAACATCAGC	21	5'Azide	97.51	97.51	1

样品	总摩尔量	Eq Oligo	Oligo 摩尔量	S1 加入量	总体积
GDH	0.5 nMol	20 Eq	10 nMol	10 μL	100 μL

注：浓度(ACr)计算公式为 (30% × 4.8) / 8

@@ Line 45: / Line 45: @@
 ! 样品 !! 原样品浓度 !! 终样品浓度 !! 加入量 !! 总体积
 |-
-|<span style="color:red;"> GDH </span>|| 0.7 mg/mL || 0.175 mg/mL || 25 μL || 100 μL
+|'''<span style="color:red;"> GDH </span>'''|| 0.7 mg/mL || 0.175 mg/mL || 25 μL || 100 μL
 |}