This technique is mainly applied to the detection of hormonal stimulants.

Operation method

Green fluorescent protein fusion technology

Principle

Recombinant human growth hormone (rhGH) and insulin growth factor-1 (ICF-I) are endogenous hormonal stimulants that are currently abused by athletes but difficult to detect. Using IGF-Ⅰ as the target analyte, molecular biology methods were used to construct GFP-IGF, a fusion protein of green fluorescent protein (GFP) and IGF-Ⅰ. GFP is a widely used bioreporter molecule, which has the characteristics of stability, non-toxicity, and self-fluorescence without substrate and cofactor, and is a very promising marker.

Materials and Instruments

Proteins
Green Fluorescent Protein Fusion Protein
PCR instrument Electrophoresis tank Fluorescence microscope

Move

1. Construct E. coli recombinant bacteria expressing fusion protein GFP-IGF by genetic recombination.

2. The fusion protein was purified and analyzed by SDS-PAGE as a single band with a molecular weight of 36KD.

3. Western blotting and ELISA were performed to verify that the fusion protein specifically reacted with IGF-1 antibody and had the antigenicity of IGF-1.

4. Surface Plasmon Resonance (SPR) analysis, obtained KD=2. ^6X10-8M for GFP-IGF with IGF-1 monoclonal antibody, which meets the requirement for immunization.

5. CD spectroscopy, fluorescence spectroscopy, and fluorescence lifetime analysis showed that the fusion protein maintained the fluorescence properties of GFP.

Caveat

1. pay attention to the specification of molecular manipulation.

2. Protein purification should be performed at low temperatures.

Common Problems

Source: Application of Green Fluorescent Protein Fusion Technology in the Detection of Hormonal Stimulants.

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