放射性脉冲追踪分析和免疫沉淀法

Javier G.  Magadán

doi:10.21769/BioProtoc.1101

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Peer-reviewed

Radioactive Pulse-Chase Analysis and Immunoprecipitation

放射性脉冲追踪分析和免疫沉淀法

Javier G. Magadán email

发布: 2014年04月20日第4卷第8期 DOI: 10.21769/BioProtoc.1101 浏览次数: 12497

评审: Anonymous reviewer(s)

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The authors used this protocol in:

Cover of Journal of Virology, featuring study using the protocol.

Sep 2013

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Abstract

Labeling of newly-synthesized polypeptides with radioactive amino acids followed by immunoprecipitation allows quantitative analysis of the fate of a given protein in a time-dependent manner. This biochemical approach is usually used to study a variety of processes, such as protein folding, co-translational modifications, intracellular transport, and even its rate of degradation. Here, I describe step by step a simple technique to both label newly-synthesized influenza A virus (IAV) hemagglutinin (HA) with [³⁵S]-methionine and then follow its maturation and transport through the secretory pathway by SDS-PAGE and fluorography (Magadan et al., 2013).

Materials and Reagents

~5 x 10⁶ MDCK cells (ATCC, catalog number: CCL-34 )
0.05% 1x Trypsin-EDTA (phenol red) (Life Technologies, catalog number: 25300054 )
DPBS (Life Technologies, catalog number: 14190-250 )
Anti-HA antibodies (mostly home-made)
nProtein A Sepharose 4 Fast Flow (GE, catalog number: 17-5280-02 )
4x NuPAGE LDS sample buffer (Life Technologies, catalog number: NP0007 )
NuPAGE Novex 4-12% Bis-Tris protein gels (Life Technologies, catalog number: NP0321PK2 )
NuPAGE MES SDS running buffer (Life Technologies, catalog number: NP000202 )
DMEM media without methionine and cysteine (Life Technologies, catalog number: 21013024 )
EDTA-free protease inhibitor cocktail (Roche Diagnostics, catalog number: 04693159001 )
10% Triton X-100 surfact-amps detergent solution (Thermo Fisher Scientific, catalog number: 28314 )
UltraPure 1 M Tris-HCI Buffer (pH 7.5) (Life Technologies, catalog number: 15567-027 )
UltraPure 5 M NaCl (Life Technologies, catalog number: 24740-011 )
UltraPure 0.5 M EDTA (pH 8.0) (Life Technologies, catalog number: 15575-020 )
Methanol (Sigma-Aldrich, catalog number: 322415 )
Acetic acid (Sigma-Aldrich, catalog number: 320099 )
DMEM media (Life Technologies, catalog number: 10569-010 )
DMEM media with 7.5% fetal bovine serum (see Recipes)
Pulse medium (see Recipes)
Chase medium (see Recipes)
Non-denaturing lysis buffer (see Recipes)
Fixation solution (see Recipes)
L-cysteine (Sigma-Aldrich, catalog number: W326305 ) (see Recipes)
[³⁵S]-methionine (PerkinElmer, catalog number: NEG-709-A005MC ) (see Recipes)
L-methionine (Sigma-Aldrich, catalog number: M9625 ) (see Recipes)

Equipment

Note: Designated for working with radioactive materials.

Appropriate containers to dispose liquid and solid [³⁵S]-waste following local radiation safety guidelines
T-75 tissue culture flask (Thermo Fisher Scientific)
50 ml BD Falcon tube (BD Biosciences, Falcon^®)
A water bath set at 37 °C
A refrigerated micro-centrifuge
A rotator for 1.5 ml micro-centrifuge tubes settled at 4 °C
1.5 ml micro-centrifuge tubes
A chamber to run mini-gels [I routinely use the XCell SureLock Mini Cell electrophoresis system (Life Technologies, catalog number: EI0001 ).]
Carestream Kodak BioMax MR films (Sigma-Aldrich, catalog number: Z350400 )
Kodak X-OMAT 2000A processor
A gel-dryer
A cassette for autoradiography