In Vitro Disassembly of the Influenza A Virus Core Using pH-Controlled Gradient Centrifugation

Özet

Source: Stauffer, S. et al. In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation. J. Vis. Exp. (2016)

This video demonstrates the pH-dependent disassembly of influenza A virus (IAV) cores using gradient ultracentrifugation. It outlines glycerol gradient preparation, ultracentrifugation, and how different pH conditions affect IAV core disassembly.

Protokol

1. Preparation of Buffers and Stock Solutions

1. Prepare MNT buffer (20 mM 2-(N-morpholino)ethanesulfonic acid (MES), 30 mM Tris and 100 mM NaCl) by dissolving 470 mg Tris Hydrochloride, 390 mg MES hydrate and 580 mg NaCl in 80 ml ddH₂O. Adjust the buffer to pH 7.4 and bring it to a final volume of 100 ml with ddH₂O.
2. Prepare three identical solutions of 500 mM MES buffer by dissolving 9.76 g MES hydrate in 80 ml dH₂O each. Adjust the buffers to pH 5.8, 5.4, and 5.0, respectively, by titrating with concentrated NaOH. Bring each buffer to a final volume of 100 ml with dH₂O.
3. Prepare two identical solutions o....

Malzemeler

Bu makalede kullanılan malzemelerin listesi

Ad	Şirket	Katalog numarası	Yorumlar
cOmplete™, EDTA-free protease inhibitor tablets	Sigma-Aldrich	11873580001	The stock solution can be stored at 2 to 8 °C for 1 to 2 weeks
Glycerol anhydrous BioChemica	AppliChem	A1123
Hydrochloric acid	Merck Millipore	100317
Long injection needle (21 G, 9 cm, bevel or blunt-end)
MES hydrate	Sigma-Aldrich	M8250
NP-40	Sigma-Aldrich	I8896	Now commercially available as IGEPAL® CA-630
pH indicator strips, pH 4.0-7.0	Merck Millipore	109542
Sodium chloride	Merck Millipore	106406
Sodiumhydroxide	Merck Millipore	106498
Steritop filter unit	Merck Millipore	SCGPT05RE
SW41 Ti, ultracentrifuge rotor set	Beckman Coulter	331336
Thinwall, Ultra-clear centrifuge tubes, 13.2 ml, 14 mm x 89 mm	Beckman Coulter	344059
Tris hydrochloride	AppliChem	A1087
X31 Influenza A virus (H3N2), egg-grown, clarified allantoic fluid	Virapur		Freshly thawed on 4 °C