Polyvinyl Alcohol can Stabilize FITC Conjugated Recombinant Annexin V for Apoptotic Cells Detection.
Annexin V
FITC
PVA
expression
glycerol
stability
trehalose
Journal
Protein and peptide letters
ISSN: 1875-5305
Titre abrégé: Protein Pept Lett
Pays: Netherlands
ID NLM: 9441434
Informations de publication
Date de publication:
2022
2022
Historique:
received:
28
02
2022
revised:
13
04
2022
accepted:
13
04
2022
pubmed:
21
6
2022
medline:
2
11
2022
entrez:
20
6
2022
Statut:
ppublish
Résumé
Annexin V, a member of calcium-dependent phospholipid-binding proteins, selectively binds to the exposed phosphatidylserine, which can be used for in vitro apoptosis detection. Simultaneous staining of cells with annexin V-fluorescein isothiocyanate (FITC) and the non-vital dye propidium iodide (PI) enables the detection of apoptotic and necrotic cells. Our study aimed to express, purify, and stabilize the recombinant annexin V. The recombinant annexin V was cloned and expressed in E. coli bacteria and was purified using Ni-IDA resin. The FITC conjugation was performed, and apoptosis detection of HaCaT cells by FITC-labeled annexin V was evaluated by flow cytometry. Then, the stability of FITC-labeled annexin in various conditions, including polyvinyl alcohol (PVA), glycerol, and trehalose, was evaluated. The results showed that annexin V was appropriately expressed and purified. After FITC conjugation, it could perfectly detect the cell death of HaCat cells in different apoptosis percentages. FITC-labeled annexin had more stability with PVA than glycerol and trehalose. It seems that PVA has an acceptable effect on FITC-labeled annexin V stability in concentrations lower than 1 mg mL
Sections du résumé
BACKGROUND
BACKGROUND
Annexin V, a member of calcium-dependent phospholipid-binding proteins, selectively binds to the exposed phosphatidylserine, which can be used for in vitro apoptosis detection. Simultaneous staining of cells with annexin V-fluorescein isothiocyanate (FITC) and the non-vital dye propidium iodide (PI) enables the detection of apoptotic and necrotic cells.
OBJECTIVE
OBJECTIVE
Our study aimed to express, purify, and stabilize the recombinant annexin V.
METHODS
METHODS
The recombinant annexin V was cloned and expressed in E. coli bacteria and was purified using Ni-IDA resin. The FITC conjugation was performed, and apoptosis detection of HaCaT cells by FITC-labeled annexin V was evaluated by flow cytometry. Then, the stability of FITC-labeled annexin in various conditions, including polyvinyl alcohol (PVA), glycerol, and trehalose, was evaluated.
RESULTS
RESULTS
The results showed that annexin V was appropriately expressed and purified. After FITC conjugation, it could perfectly detect the cell death of HaCat cells in different apoptosis percentages. FITC-labeled annexin had more stability with PVA than glycerol and trehalose.
CONCLUSION
CONCLUSIONS
It seems that PVA has an acceptable effect on FITC-labeled annexin V stability in concentrations lower than 1 mg mL
Identifiants
pubmed: 35718962
pii: PPL-EPUB-124602
doi: 10.2174/0929866529666220617153809
doi:
Substances chimiques
Annexin A5
0
Fluorescein
TPY09G7XIR
Fluorescein-5-isothiocyanate
I223NX31W9
Polyvinyl Alcohol
9002-89-5
Trehalose
B8WCK70T7I
Glycerol
PDC6A3C0OX
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
806-814Subventions
Organisme : Research Administration Department of Mashhad University of Medical Sciences, Iran
ID : 971748
Informations de copyright
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