Team:NYMU-Taipei/Experiments

TEAM

Team Members

Collaborations

Attributions

PROJECT

Description

Design

Protein Expression

Immobilization

Application

Demonstrate

AWARDS

Hardware

Measurement

Model

PARTS

Parts Overview

Basic Parts

Composite Parts

Parts Collection

Improve

HUMAN PRACTICES

Human Practices

Education & Engagement

SAFETY

JUDGING FORM

Experiments & Results

• Week 1
• Week 2
• Week 3
• Week 4
• Week 5

Immobilization

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In order to popularize the examination to the public, we need to immobilize the protein on some low-cost materials which can be seen everywhere. After looking up the concerned information, we found three ways which might be feasible in our project. Two of them are for immobilization on paper, and the other use plastic surface(PP) as the support.

Immobilization on Paper with Chitosan and Glutaraldehyde

Material

• 85% deacetylation chitosan
• 2.5% glutaraldehyde in 0.01 mol/L, pH 7.4 PBS
• Whatman chromatography paper#1 (WCP#1) (pure cellulose paper)
• Ultrapure water
• Blotting paper
• Coupling buffer: 0.01 mol/L pH 7.4 phosphate buffer solution (PBS)
• Blocking buffer: PBS containing 0.5% BSA and 0.5% casein
• Wash buffer: 0.05% Tween-20 was spiked into 0.01 mol/L pH7.4 PBS (PBST)

Protocol

1. 5.0 µL of 0.25 mg/mL chitosan were dropped into the paper and dried at room temperature.
2. Let the paper activate with 2.5% glutaraldehyde for 2 h.
3. Then the paper was washed twice by adding 20 µL of ultrapure on the paper.
4. Using the blotting paper to absorb the excess wash water.
5. 4 µL of 30 µg/mL proteins in coupling buffer were applied to the paper and activating for 30 min at room temperature.
6. Then, 20 µL of ultrapure water were used to wash excess protein.
7. 20 µL blocking buffer was applied to the paper and activated for 15 min at room temperature.
8. Then, the protein modified paper zones were washed twice with washing buffer.

Immobilization on Paper by Periodate oxidation

Material

• 1.6 mg/mL NaCNBH₃
• 0.5 mol/L NaIO₄
• bovine serum albumin(BSA)
• Whatman chromatography paper#1 (WCP#1) (pure cellulose paper)
• Ultrapure water
• Blotting paper
• Coupling buffer: 0.01 mol/L pH 7.4 phosphate buffer solution (PBS)
• Blocking buffer: PBS containing 0.5% BSA and 0.5% casein
• Wash buffer: 0.05% Tween-20 was spiked into 0.01 mol/L pH7.4 PBS (PBST)

Protocol

1. 10 mL of 0.5 mol/L NaIO₄ was spotted on the paper, and react at room temperature in the dark for 30min.
2. The paper were washed twice by adding 10 mL of ultrapure water, and then use the blotting paper to absorb the excess water.
3. 3 mL of proteins (80 mg/mL) in coupling buffer were added to the paper, and incubating for 30 minutes at room temperature.
4. Excess antibodies were washed with 10 mL of ultrapure water.
5. Treated the paper with 10 mL 1.6 mg/mL NaCNBH₃
6. Adding 10 mL of blocking buffer and dry for 15 min under ambient conditions.
7. Then washed twice with 10 mL of washing buffer.