Gm1 ganglioside to annexin v microparticle polypeptide ratio for biological monitoring
A ganglioside and annexin technology, applied in biological testing, biochemical equipment and methods, microbial measurement/testing, etc., can solve problems such as high heterogeneity
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[0164] Microparticle preparation
[0165] CTB or Annexin V-bound microparticles can be prepared from plasma (or other fluid) by the following protocol:
[0166] 10 μL plasma was treated with 0.1 μg biotinylated cholera toxin subunit B (CTB) (SBL Vaccin AB) or 0.1 μg biotinylated annexin V (AV) (BioVision) in 100 μL, pH 7.4 PBS at Incubate at 37°C for 1 hour with shaking at 800 rpm. At the same time, 100 μL M-280 streptavidin (Invitrogen) was washed three times with 100 μL PBS. After the last wash, the plasma-CTB or plasma-AV reaction mixture was added to the washed beads and incubated for 30 min with shaking at 800 rpm. The beads were fixed with a magnet and the supernatant was removed. The beads were then washed three times with 200 μl of PBS, and the eluate was removed after each fixation of the beads with a magnet.
[0167] Ratio of GM1 ganglioside microparticle polypeptide to annexin V microparticle polypeptide
[0168] The ratio of GM1 ganglioside particle polypept...
Embodiment 1
[0174] Example 1: Enrichment of CD9+ microparticles from serum of normal individuals
[0175] In this study, the inventors determined for the first time whether CTB binds to CD9+ microparticles in serum. Due to the lack of CTB-bound CD9, the inventors determined the sedimentation density of serum CD9+ microparticles in a sucrose density gradient and found that the sedimentation density of CD9 was higher in serum than in exosomes.
[0176] The lack of CTB binding and the high sedimentation density suggest that CD9+ microparticles are apoptotic bodies. This was subsequently confirmed by the binding of serum CD9 to Annexin V.
Embodiment 2
[0177] Example 2: Materials and Methods - Molecular Sieve Chromatography
[0178] 2mL 2B resin (Sigma Aldrich, Cat no. 2B300) was packed into a spin column (Bio-Rad, Cat no. 732-6008) and centrifuged at 800 g for 1 min. The supernatant was removed and rinsed by adding 1 mL of phosphate-buffered saline (PBS), followed by centrifugation at 800 g for 1 min. Subsequently, 1 mL of human serum was loaded and centrifuged at 800 g for 1 min.
[0179] The flow through was collected and the process was repeated three times with 1 mL of PBS each time. Three eluate fractions were collected. Take 20 μl of each fraction and separate on 4-12% SDS / polyacrylamide gel. The resulting gel was processed with SilverQuest TM Silver Staining Kit (Invitrogen, Carlsbad, CA) staining, or electrotransfection onto nitrocellulose membranes. The membrane was labeled with mouse anti-human CD9 antibody diluted 1:50, followed by HRP-conjugated donkey anti-mouse IgG antibody diluted 1:1250.
[0180] Al...
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