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Protein & Peptide Letters

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ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

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Research Article

Polyvinyl Alcohol can Stabilize FITC Conjugated Recombinant Annexin V for Apoptotic Cells Detection

Author(s): Saeideh Sadat Shobeiri and Mojtaba Sankian*

Volume 29, Issue 9, 2022

Published on: 08 September, 2022

Page: [806 - 814] Pages: 9

DOI: 10.2174/0929866529666220617153809

Price: $65

Abstract

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: Our study aimed to express, purify, and stabilize the recombinant annexin V.

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: 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: It seems that PVA has an acceptable effect on FITC-labeled annexin V stability in concentrations lower than 1 mg mL-1 without interfering with fluorescent intensity.

Keywords: Annexin V, expression, FITC, stability, PVA, glycerol, trehalose.

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