Protein cross-linked nano-silicon and preparation method, exosome separation and purification method and application
A protein cross-linking, separation and purification technology, applied in biochemical equipment and methods, cell dissociation methods, microorganisms, etc., can solve the problems of time-consuming and labor-consuming, low purity of exosomes, high cost, etc., to save time and The effect of cost, high purity, and guaranteed quality
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Embodiment a
[0062] Example A: 40ml of the outer bude liquid added to 10 6 10 7 10 8 10 9 10 10 Particles Number of Annexin V Crosslinked 10 μm Nanoicon (Set to Annexin V A, Annexin VD, Annexin V, Annexin VD, Annexin V E Series), add CACL 2 By 15 mm (millimoles per liter), the lower speed is mixed with 50 mL, and the shaker is mixed onto the shaker at a low speed of the shaker at 4 ° C; centrifuge at 2000g, abandon the supernatant and collect the precipitate; 1 ml CA- HEPESBuffer (including 10mm hepes, pH 7.5; 1mm MGCL 2 ; 5mm KCl; 2% BSA; 15mm CaCl 2 And 150 mMNacl was washed 3 times and naturally dried; then 500 ul eluent (containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA) was mixed with precipitate for 10 min, 4 ° C. Cartridge, centrifugation, The supernatant was collected, that is, the purified concentrated exosomer; the obtained exocial body was stored in the -80 ° C refrigerator.
Embodiment b
[0063] Example B: 40M Extracedrous Extraction of 10 6 10 7 10 8 10 9 10 10 Particles number of TIM 4 crosslinked 10 μm nano silicon (set to TIM 4A, TIM 4B, TIM 4C, TIM 4D, TIM 4E series), add CACL 2 To 2 mm, the shaker is mixed with 50 mL, and the lower speed is mixed with the shaker at low speed to the underside of 4 ° C. The post 4 ° C was centrifuged for 10 min, discarded the supernatant and collected precipitation; with washing disengagement (comprising 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.0005% Tween 20, 2mmcacl) 2 Centrifugation was washed 3 times and naturally air dried, 500 ul (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mMedta) was added to 500 ul eluent (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mm EDTA) and precipitation The mixture was mixed for 10 min, 4 ° C. Centrifuge at 4 ° C for 10 min, and the supernatant was collected, that is, the purified outer body; the obtained exocrid was prepared in the -80 ° C refrigerator.
[0064] Through NTA detection, the number of outer bo...
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