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Purification of 38kDa Antigen of Mycobacterium tuberculosis by Two Dimensional Preparative Electrophoresis

采用二维双向电泳法纯化结核杆菌的38kDa抗原

Uma Devi Ranganathan Uma Devi Ranganathan

发布: 2012年12月20日第2卷第24期 DOI: 10.21769/BioProtoc.312 浏览次数: 10840

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Cover of Tuberculosis, featuring study using the protocol.

Jun 2001

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Abstract

Serodiagnosis of tuberculosis using purified native antigens is one of the approach for the early detection of TB. This protocol gives the details for growing Mycobacterium tuberculosis in liquid culture and one proven way to get a good yield of the total secreted antigens in the culture supernatant. In addition the procedure for purification of the 38 kDa antigen from the total culture filtrate antigens is also explained. 38 kDa is one of the specific antigen for Mycobacterium tuberculosis complex and in this particular study it was used as one of the antigens for serodiagnosis of different category of tuberculosis patients.

Keywords: Preperative Electrophorosis (制备电泳) search Rotophor (Rotophor) search 38kDa antigen of Mycobacterium tuberculosis (结核分枝杆菌38KDa蛋白抗原) search Antigens of TB (结核抗原) search Two dimenstional preparative electrophoresis (二维电泳) search

Materials and Reagents

  1. Mycobacterium tuberculosis (M tuberculosis) H37Rv
  2. Glycerol
  3. Potassium dihydrogen-orthophosphate (anhydrous)
  4. Magnesium sulphate (7H2O)
  5. Citric acid
  6. Ferric ammonium sulphate
  7. Sodium glutamate
  8. Casein hydrolysate
  9. Milli Q water
  10. Sodium azide
  11. BCA assay kit (Pierce Antibodies, catalog number: 23252 )
  12. Protease inhibitor (Roche Diagnostics, catalog number: 05-056-489-001 )
  13. Phenyl methyl sulfonyl fluoride (PMSF)
  14. Urea
  15. Digitonin (Sigma-Aldrich, catalog number: D141-100MG )
  16. CHAPS
  17. Carrier ampholytes
  18. 6x SDS sample buffer
  19. Glass beads
  20. Protein dialysis bag (Thermo Fisher Scientific, catalog number: 88244 )
  21. McCartney bottle
  22. Millipore filter (0.22 micron) (EMD Millipore, catalog number: SLAP02550 )
  23. Sauton's liquid medium (see Recipes)

Equipment

  1. Centrifuges
  2. Magnetic stirrer
  3. Preparatory Preparative isoelectric focusing (IEF) cell (Bio-Rad Laboratories, Rotofor, catalog number: 170-2906 )
  4. Model 491 Prep Cell (Bio-Rad Laboratories, catalog number: 170-2926 )
  5. Bacterial incubator
  6. Protein concentrator 3 kDa cut-off-stirred cell (Life Technologies, Ambion®, catalog number: 5123 )
  7. Circulating water bath
  8. 3 mm single glass beads

Procedure

English
中文翻译

文章信息

版权信息

© 2012 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Ranganathan, U. D. (2012). Purification of 38kDa Antigen of Mycobacterium tuberculosis by Two Dimensional Preparative Electrophoresis. Bio-protocol 2(24): e312. DOI: 10.21769/BioProtoc.312.

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分类

微生物学 > 微生物生物化学 > 蛋白质 > 分离和纯化

生物化学 > 蛋白质 > 分离和纯化

生物化学 > 蛋白质 > 电泳

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