Zonal Sedimentation Analysis on Sucrose Gradients
蔗糖密度梯度分区沉淀分析
发布: 2014年04月20日第4卷第8期 DOI: 10.21769/BioProtoc.1100 浏览次数: 9892
评审: Anonymous reviewer(s)
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The authors used this protocol in:
Sep 2013
Abstract
Zonal sedimentation analysis on sucrose gradients allows estimation of the molecular size of an individual protein or a protein complex by centrifugation at a constant speed under nondenaturing conditions. This method is particularly suitable for globular proteins like the influenza A virus (IAV) protein hemagglutinin (HA). Here, I describe step by step a protocol used to evaluate the oligomeric state of recombinant HA trimers (Magadan et al., 2013).
Materials and Reagents
- Trimerized recombinant HA (recHA3) derived from influenza A/Puerto Rico/8/34 (PR8) virus (Magadan et al., 2013)
- Gel filtration protein standards [carbonic anhydrase (29 kDa), ovalbumin (43 kDa), conalbumin (75 kDa), aldolase (158 kDa), and ferritin (440 kDa)] (GE, catalog numbers: 28-4038-41 and 28-4038-42 )
- 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 )
- Ponceau S solution (Sigma-Aldrich, catalog number: P7170 )
- 5% acetic acid
- Blotting grade blocker nonfat dry milk (Bio-Rad Laboratories, catalog number: 170-6404XTU )
- 1x PBS (Life Technologies, catalog number: AM9624 )
- Tween-20 (Sigma-Aldrich, catalog number: P1379 )
- A home-made, conformation-independent mouse monoclonal antibody to denatured HA1 (clone CM-1)
- A rabbit polyclonal anti-mouse HRP-conjugated antibody (Dako, catalog number: P0260 )
- SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, catalog number: 34077 )
- High purity sucrose (Thermo Fisher Scientific, catalog number: NC0110097 )
- UltraPure 5 M NaCl (Life Technologies, catalog number: 24740-011 )
- UltraPure 0.5 M EDTA (pH 8.0) (Life Technologies, catalog number: 15575-020 )
- Sucrose gradient (see Recipes)
Equipment
- Refractometer (Bausch & Lomb Incorporated)
- 14 x 89 mm Ultra Clear tubes (Beckman Coulter, catalog number: 344059 )
- Pipettor
- 1.5 ml micro-centrifuge tubes
- An ultracentrifuge equipped with a SW41 rotor (Beckman Coulter)
- Nitrocellulose blotting membranes (0.45 µm pore size) (Life Technologies, catalog number: LC2000 )
- A chamber to run mini-gels [I routinely use the XCell SureLock Mini Cell electrophoresis system (Life Technologies, catalog number: EI0001 ).]
- A Mini Trans-Blot Cell (Bio-Rad Laboratories, catalog number: 170-3930 )
- Carestream Kodak BioMax XAR films (Sigma-Aldrich, catalog number: F5388 )
- Kodak X-OMAT 2000A processor or equivalent
- A cassette for autoradiography
Software
- ImageJ software (http://imagej.nih.gov/ij/)
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Magadán, J. G. (2014). Zonal Sedimentation Analysis on Sucrose Gradients. Bio-protocol 4(8): e1100. DOI: 10.21769/BioProtoc.1100.
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分类
微生物学 > 微生物生物化学 > 蛋白质
生物化学 > 蛋白质 > 结构
生物化学 > 蛋白质 > 电泳
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