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| <div style="color: black !important; font-family: 'Times New Roman', SimSun, serif !important, line-height:1.5;">
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| == Protein(RuCHMO/GDH) Conjugation to DNA Origami ==
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| === Methods ===
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| === Conjugation ===
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| *''' 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.'''
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|
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| === Incubation ===
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| *'''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 ℃.'''
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| === Oligonucleotide Sequence ===
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| *'''sequence(RuCHMO):CTCTCCTTCTTCCCTTTCTTT'''
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| *'''Sequence(GDH):TTCGACAGCATGAACATCAGC'''
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| === Protein ===
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| *'''RuCHMO: 65.45 KDa'''
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| *'''GDH: 35 KDa'''
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|
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| === Purification ===
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| *''' 30 kDa MWCO 0.5 ml Amicon centrifugal filters (Milli-pore)'''
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| === electro-phoresis ===
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| {| class="wikitable"
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| |+ Sample
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| |-
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| ! Reagent
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| ! Sequence
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| |-
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| | DBCO-PEG4-Bis-sulfone
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| |-
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| | azide modified oligonucleotide
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| | CTCTCCTTCTTCCCTTTCTTT
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| |-
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| | azide modified oligonucleotide
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| | TTCGACAGCATGAACATCAGC
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| |}
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| <div style="color: black !important; font-family: 'Times New Roman', SimSun, serif !important, line-height:1.5;">
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|
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| == Protocol (版本 1.0) ==
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| === 1. GDH 制备 ===
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| 取 '''17.5 μg GDH''' 溶解于 '''25 μL 1×PBS''' 中。
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|
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| === 2. 稀释 GDH（样品液） ===
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| {| class="wikitable" style="text-align:center; width:80%"
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| ! 样品 !! 原样品浓度 !! 终样品浓度 !! 加入量 !! 总体积
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| |-
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| | GDH || 0.7 mg/mL || 0.175 mg/mL || 25 μL || 100 μL
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| |}
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|
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| === 3. 溶解 DBCO-PEG4-Bis-Sulfone 于 DMF(N,N-二甲基甲酰胺) 中（100 mM） ===
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|
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| === 4. 稀释 DBCO（样品液） ===
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| {| class="wikitable" style="text-align:center; width:80%"
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| ! 样品 !! 原样品浓度 !! 终样品浓度 !! 加入量 !! 总体积
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| |-
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| | DBCO || 100 mM || 20 mM || 2 μL || 10 μL
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| |}
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|
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|
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| === 5. 向 GDH 样品液中加入 DBCO，并在室温下孵育 4 h ===
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| {| class="wikitable" style="text-align:center; width:90%"
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| ! 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
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| |-
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| | GDH || 5 mM || 35 kDa || 0.5 nMol || 20 Eq || 10 nMol || 0.5 μL || 100 μL
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| |}
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|
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| === 6. 使用 10 kDa 超滤管超滤去除游离 DBCO ===
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|
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|
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| === 7. 加入 Oligo，并在 37 ℃ 下孵育 4 h ===
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| {| class="wikitable" style="text-align:center; width:90%"
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| ! 样品 !! 总摩尔量 !! Eq Oligo !! Oligo 摩尔量 !! Oligo 加入量 !! 总体积
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| |-
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| | GDH || 0.5 nMol || 20 Eq || 10 nMol || 0.5 μL || 100 μL
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| |}
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|
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| === 8. 凝胶电泳 ===
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| 等量的 '''GDH''' , '''偶联产物''' 和 '''Marker''' 跑胶，对蛋白进行'''考马斯亮蓝'''染色比对。
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