Preparation and application of test paper for testing royal jelly freshness
A technology for testing test strips and royal jelly, applied in biological testing, measuring devices, material inspection products, etc., can solve problems such as loss of functional components, judging the freshness and quality of royal jelly, and deterioration.
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Embodiment 1
[0165] Two-dimensional electrophoresis separation and mass spectrometry analysis of royal jelly protein
[0166] The royal jelly under different storage conditions was extracted by phosphoric acid extraction buffer (PB) and protein sample dissolution buffer (LB), respectively, and trichloroacetic acid was added to the extract to precipitate the protein, and the whole protein of royal jelly under different storage conditions was obtained. The protein is redissolved and the concentration is detected, then two-dimensional electrophoresis (solid-phase pH gradient, 17cm gel strip, pH3-10) is performed according to a certain loading amount, and the electrophoretic spectrum ( figure 1 ) is analyzed with professional analysis software. The target protein spots with high expression abundance were digested for mass spectrometry analysis, and the mass spectrometry results were submitted to a professional database for sequence comparison for protein identification ( figure 2 ). The ana...
Embodiment 2
[0168] Chromatographic Separation and Mass Spectrometry Analysis of Royal Jelly Protein
[0169] Royal jelly stored under different storage conditions was precipitated with acetone and redissolved in alkaline buffer (pH8.2) to obtain the total protein of fresh royal jelly, royal jelly stored at -20°C for one year, royal jelly stored at 4°C for one year, and royal jelly stored at room temperature for one year , the above protein samples were subjected to molecular sieve chromatographic separation ( image 3 ), it is determined that there are significant differences in the components with molecular weights from 10K to 100K. The fourth fraction (Re=17.34min) was selected and subjected to trypsin digestion, sodium upgrading chromatographic separation and mass spectrometry sequence analysis (Nano LC MS / MS, Table 1), and it was confirmed that royal jelly protein 5 can only be stored in fresh royal jelly and at -20°C detected in royal jelly.
Embodiment 3
[0171] Detect target protein gene clone expression
[0172] The respective forward and reverse primers were synthesized according to the sequences of apal jelly protein 4, apal jelly protein 5 and glucose oxidase provided in the NCBI database, and PCR amplification was carried out using the DNA of the head of the honeybee worker bee as a template. Connect the amplified target fragment with an appropriate carrier (T plasmid, kit) to form a recombinant, and then introduce the recipient bacteria (E. Nucleotide sequence analysis. The recombinants confirmed by sequencing were recombined into expression vectors and induced to express them. After the expression product was isolated and purified, it was detected by mass spectrometry.
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