Lectin Binding Analysis of Streptococcus mutans Glycoproteins
变异链球菌糖蛋白的凝集素结合分析
发布: 2015年04月05日第5卷第7期 DOI: 10.21769/BioProtoc.1431 浏览次数: 8891
评审: Anonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Aug 2014
Abstract
Bacterial glycoproteins are of increasing interest due to their abundance in nature and importance in health and infectious diseases. However, only a very small fraction of bacterial glycoproteins have been characterized and its post-translational modification machinery identified. While analysis of glycoproteins can be achieved through various techniques, this is often limited by the specific characteristics of individual proteins such as type and level of glycosylation. Lectins are sugar-binding proteins that recognize specific glycoconjugates in a manner similar to antigen-antibody interactions. Here, we describe a simple method for the detection of glycoproteins using lectin-based Western blot analysis, which can be applied to different organisms and coupled with various other strategies for complementary analysis.
Materials and Reagents
- Bacterial whole cell lysates
- Brain heart infusion medium (BHI) (BD Biosciences, catalog number: 237500 )
- Biotinylated lectin [Vector Laboratories, for Wheat Germ Agglutinin (WGA), catalog number: B-1025]
- HRP-conjugated streptavidin (Cell Signaling Technology, catalog number: 3999 )
- Tween 20 (Sigma-Aldrich, catalog number: P2287 )
- Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418 )
- Enhanced chemiluminiscent detection kit (GE Healthcare, catalog number: RPN2108 )
- Autoradiography films (Carestream Health, catalog number: 864-6770 )
- NaCl (J.T.Baker®, catalog number: 3628-01 )
- KCl (J.T.Baker®, catalog number: 3045-01 )
- Na2HPO4 (J.T.Baker®, catalog number: 3827-01 )
- KH2PO4 (J.T.Baker®, catalog number: 3246-01 )
- Tris base (J.T.Baker®, catalog number: 4109-02 )
- Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
- Glycine (Thermo Fisher Scientific, catalog number: BP381-1 )
- 30% Acrylamide/Bis solution (Bio-Rad Laboratories, catalog number: 161-0158 )
- Tetramethylethylenediamine (TEMED) (Life Technologies, catalog number: 15524-010 )
- Ammonium persulfate (Bio-Rad Laboratories, catalog number: BP179-25 )
- Glycerol (Alfa Aesar, catalog number: A16205 )
- Methanol (BDH Chemicals, catalog number: BDH1135-4LP )
- Bromophenol blue (Sigma-Aldrich, catalog number: B5525 )
- 1x phosphate buffer solution (PBS) (see Recipes)
- Resolving gel (see Recipes)
- Stacking gel (see Recipes)
- 2x sample buffer (see Recipes)
- Running buffer (see Recipes)
- Transfer buffer (see Recipes)
- Blocking solution (see Recipes)
- Primary solution (see Recipes)
- Secondary solution (see Recipes)
- Washing solution (see Recipes)
Equipment
- 0.1 mm glass beads (Research Products International)
- Polyvinylidene fluoride (PVDF) membranes (EMD Millipore, catalog number: P2563-10EA )
- 3 mm Whatman paper (GE Healthcare)
- CO2 Incubator
- Sterile culture tubes (15 ml)
- Screw cap tubes (1.7 ml)
- Bead beater (BioSpec Products)
- Protein electrophoresis running apparatus
- Protein transfer apparatus
- Power supply
- Rocker
- Film developer
- pH meter
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Avilés-Reyes, A., Lemos, J. A. and Abranches, J. (2015). Lectin Binding Analysis of Streptococcus mutans Glycoproteins. Bio-protocol 5(7): e1431. DOI: 10.21769/BioProtoc.1431.
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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 > 免疫检测
生物化学 > 糖类 > 糖蛋白
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