XIE/Enzyme:2025/Dec: Difference between revisions

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#琼脂糖凝胶电泳
*琼脂糖凝胶电泳
[[File:Origami_Enzyme_Success.png|thumb|center|center|600px]]
[[File:Origami_Enzyme_Success.png|thumb|center|center|600px]]

Revision as of 13:40, 28 December 2025

12.08

  • HR-DNA Origami（NO） Folding
  • Materials
Material Volume（100 μL）
Staples (500 nM) 25 μL
P8064 (150 nM) 10 μL
10X PBS 10 μL
H2O 55 μL
  • Program folding in PCR thermocycler : The mixed sample was put on a thermal ramp starting with a rapid heat denaturation at 80 °C for 5 min followed by cooling from 80 °C to 60 °C over 20 min, then slow cooling from 60 °C to 24 °C over 14 h.
  • Purification (12.09): Reserve a 10 µL aliquot. Add the remaining 90 µL to a 100‑kDa MWCO centrifugal filter unit and dilute to 500 µL with 1× PBS. Centrifuge at 8,000 g for 1.5 min and discard the filtrate . Add UP Water to 500 ul to the filter unit and repeat the centrifugation; perform this wash step three times. After the final wash, adjust the volume in the filter unit to 100 µL with UP Water, invert the filter unit into a fresh collection tube, and centrifuge at 8,000 g for 1.5 min to collect the concentrate.

12.09

  • HR-DNA Origami（NO） Folding
  • Materials
Material Volume（100 μL）
Staples (490 nM) 25 μL
P8064 (150 nM) 10 μL
10X PBS 10 μL
H2O 55 μL
  • Program folding in PCR thermocycler : The mixed sample was put on a thermal ramp starting with a rapid heat denaturation at 80 °C for 5 min followed by cooling from 80 °C to 60 °C over 20 min, then slow cooling from 60 °C to 24 °C over 14 h.
  • Purification (12.10): Reserve a 10 µL aliquot. Add the remaining 90 µL to a 100‑kDa MWCO centrifugal filter unit and dilute to 500 µL with 1× PBS. Centrifuge at 8,000 g for 1.5 min and discard the filtrate . Add 1X PBS to 500 ul to the filter unit and repeat the centrifugation; perform this wash step three times. After the final wash, adjust the volume in the filter unit to 100 µL with 1X PBS, invert the filter unit into a fresh collection tube, and centrifuge at 8,000 g for 1.5 min to collect the concentrate.

12.10

  • Agarose Gel Electrophoresis
  • Gel Preparation
  1. Recipe: 2.4 g agarose in 120 mL 0.5× TBE
  2. Heat in a microwave to dissolve, cool slightly, adjust volume with 0.5× TBE to 120 ul, then add 1.2 mL of 1 M MgCl2 and 12 µL GoldView DNA stain.
  3. Insert comb and waite for the gel to cool and solidify.
  • Sample preparation
  1. Origami (15 nM): sample:loading buffer = 5:1.
  2. Scaffold: take 1 µL scaffold, dilute to 10 µL (final concentration 15 nM); scaffold:loading buffer = 5:1.
  • Electrophoresis
  1. Run at 90 V for 120 min in 0.5× TBE with 10 mM MgCl2
  2. Marker、Scaffold（1X）、Origami（10.08-未洗）、Origami（10.08—清洗）、Origami（10.09-未洗）、Origami（10.09-清洗）、Scaffold（10X）、Marker
Origami(NO)

12.11

  • TEM

Negative staining (uranyl acetate)

  • Grid activation: glow‑discharge copper grids for 30 s.
  • Sample staining
  1. apply 4.5 µL of sample onto the grid.
  2. Blot off the sample, add buffer to the grid, then blot off the buffer.
  3. Prepare parafilm and place three drops of uranyl acetate (pH adjusted) on the parafilm.
  4. Quickly transfer the grid into a staining droplet, blot off excess, and incubate for 30 s.
  5. Air‑dry the grid.
TEM_Origami(NO)

12.12

  • S2 conjugated G5

Stock Solution: 1 mM

Incubation

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

Native PAGE

  • Gel: 7.5% native gel.
  • Sample preparation: Mix 16 μL of each sample (C, M, N-U, U) with 4 μL loading buffer and mix thoroughly.
  • Electrophoresis: Run at 120 V for 100 min in 1× TBE.

12.13

  • S2 conjugated G5

Incubation

  1. Prepare two aliquots of G5 stock, labeled A and B.
  2. Add 1 µL DBCO to each of A and B and incubate for 4 h.
  3. For A: remove 16 µL as sample M1; add 10 µL S2 to the remaining A (named O1) and incubate for 4 h.
  4. For B: dilute the sample to 500 µL, transfer to a 10 kDa centrifugal ultrafiltration tube and centrifuge at 10,000 g for 30 min; concentrate to 100 µL, remove 16 µL as sample M2; add 10 µL S2 to the remaining B (named O2) and incubate for 4 h

Nativepage

  • Gel:4% stacking gel / 10% resolving gel.
  • Sample preparation: Mix 16 μL of each sample (C, A, M, B) with 4 μL loading buffer and mix thoroughly.
  • Electrophoresis: Stacking Gel:75 V; Resolving Gel:100 V.1X TBE

12.15

  • S2 conjugated enzyme

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:
  1. 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.)
  2. 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:
  1. Mix 16 µL of sample with 4 µL loading buffer, vortex, and boil at 100 °C for 10 min to inactivate.
  2. 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.
5 Enzyme

12.17

  • S2 conjugated enzyme

Sample preparation

  • Bis-Sulfone-PEG4-DBCO;取DBCO存储液 10 ul,先加入60 ul DMF，再加入 30 ul 1X PBS(7.5)，作为工作液（1 mM,30% 水）在 37℃孵育 1.5 h.
  • Al、SS、RA:取Al、SS、RA 存储液，各用 1X PBS（pH 6.5）稀释至 240 ul(5 uM),各留样 30 ul 作为阴性对照，剩余作为工作液。

Reaction

AL/ul AL/ul SS/ul SS/ul RA/ul RA/ul
Enzyme 105 105 105 105 105 105
DBCO 5 (10:1) 15 (30:1) 10 (20:1) 15 (30:1) 5 (10:1) 10 (20:1)
PBS (7.0) 10 0 5 0 10 5
Incubation / 5 h on ice on ice on ice on ice on ice on ice
SsDNA S1 5 (10:1) S1 15 (30:1) S1 10 (20:1) S2 15 (30:1) S1 5 (10:1) S1 10 (20:1)
Incubation / overnight On ice On ice On ice On ice On ice On ice

SDS page

  • Gel:10% concentration
  • Running Buffer:Tris:3.03g、Glycine:14.4g、SDS:1g、Water:1 L.
  • Stain:Coomassie blue 37℃ Sway
  • Sample:
  1. Mix 16 µL of sample with 4 µL loading buffer, vortex, and boil at 100 °C for 10 min to inactivate.
  2. Marker, C-A, M-A5, A5, M-A15, A15, C-S, M-S10, S10, M-S15, S15.
  3. Marker, C-R, M-R5, R5, M-R10, R10
Al,SS
RA

12.26

  • Step one
  1. 取 2 ul DBCO 用 6 ul PBS（pH 7.5）和 12 ul DMF 稀释至 1 mM，37℃孵育1.5 h.
  2. 将 GDH、G5、Al、RH 放在冰上等待融化，留样 16 ul作为 C 组（C-GDH、C-G5、C-Al、C-RH）；保证各样品为 116 ul 5uM.
  3. 向四个样品中各加入 5 ul 的孵育好的DBCO，在冰上 4 ℃孵育5 h.
  • 制备Origami
  1. 混合二号位点3’处用S1’修饰的Staples(2-3'-1')--用来连接 S1-G5、S1-Al、S1-RH
  2. 混合一号位点3’处用S2’修饰的Staples(1-3'-2')--用来连接 S2-GDH
  3. 混合二号位点3’处 用S1’和 一号位点3’处用S2’修饰的Staples(2-3'-1'& 1-3'-2')--用来连接 S2-GDH & S1-G5； S2-GDH & S1-Al； S2-GDH & S1-RH.
  4. 制备Origami（NO）10 ul X 1;Origami(2-3'-1') 10 ul X 3;Origami(1-3'-2') 10 ul X 1;Origami(2-3'-1'& 1-3'-2') 10 ul X 3
  5. PCR 程序孵育
  • Step two
  1. 分别留样孵育好的四个酶 16 ul,，作为 M 组（M-GDH、M-G5、M-Al、M-RH）；向 GDH 样品中加入 5 ul S2，向 G5、Al、RH 样品中加入 5 ul S1，冰上 4 ℃孵育过夜

12.27

  • step four
  1. 将孵育好的四个酶，取样 16 ul作为样品组（GDH、G5、Al、RH）
  2. 将 C、M、样品组跑SDS page(10%)
  • Incubation
  1. 将四个酶样品用 1 X PBS（pH 7.0）洗3次
  2. 将酶和Origami混合 PCR孵育 4 h
Origami (NO) O-GDH O-G5 O-AI O-RH O-GDH-G5 O-GDH-Almost O-GDH-RH
Origami/ul 10 10 10 10 10 10 10 10
Enzyme/ul 0 3 3 3 3 6 6 6
PBS/ul 6 3 3 3 3 0 0 0
  • 琼脂糖凝胶电泳
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