Preparative disk gel electrophoresis for antigen preparation

icon Preparative disk gel electrophoresis for antigen preparation

　Proteins from mammals and plants are often overexpressed as inclusion bodies in Escherichia coli. When specific antibodies are prepared, antigens should be purified from the inclusion bodies. In a simple method for antigen preparation, inclusion bodies are often washed with detergent-containing buffers such as Triton X-100 (ref. 1). However, sometimes when recombinant proteins are expressed in E. coli at low levels, the purity of the target protein is not enough. In such cases, further purification of inclusion bodies by preparative disk gel electrophoresis is a useful method forantigen preparation (ref. 2).
　Preparative disk gel electrophoresis has several advantages in antigen preparation from inclusion bodies.
  1. Preparative disk gel electrophoresis can be used to fractionate a target protein with high resolution (See Experimental examples).
  2. A target protein purified from E. coli inclusion body is recovered in soluble form in SDS electrode buffer. This purified protein can be directly injected as an antigen to elicit antibody production, without any subsequent protein concentration step.
  3. Several tens of milligrams of protein can be separated on one disk gel. This purification capacity of the disk gel column is sufficient for purification of antigen to produce rabbit polyclonal antibodies.
  4. We use the system mentioned below(※). This system is easy to operate because it uses the electroosmotic medium pump, instead of the electronic peristaltic pump used by other preparative disk gel electrophoresis systems such as the Model 491 Prep Cell (Bio-rad). Moreover, the apparatus is inexpensive.　　　　　　　　　　　　　　　　　　　　　　　　　　 ※Preparative disk gel electrophoresis system (Nihon eido, NA-1800)
 ref. 1)　　Harlow, E. and Lane, D.,
　　 　　 　　 　　Antibodies: a laboratory manual (1988), CSHL press.
 ref. 2)　　Okegawa, Y., Koshino, M., Okushima, T. and Motohashi, K.,
　　 　　 　　 　　 Protein Expr. Purif. vol.118, 77-82 (2016)
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More information is provided in the following references.
 ref.2　　Yuki Okegawa, Masanori Koshino, Teruya Okushima and Ken Motohashi 
　　　　　"Application of preparative disk gel electrophoresis for antigen purification
　　　　　　　　from inclusion bodies"
　　　　 　Protein Expr. Purif. 118, 77-82 (2016)
 
 Experimental example 1)
・Acetyl-CoA carboxylase (~50 kDa)
・Acetyl-CoA carboxylase could be purified from inclusion bodies washed with Triton X-100. Contaminat proteins from E. coli were removed by preparative disk gel electrophoresis.
・A specific Acetyl-CoA carboxylase antibody could be produced using antigen purified by this method (ref. 2).
 
 Experimental example 2)
・~15 kDa.
・E. coli contaminant proteins were removed from the 15 kDa target protein by preparative disk gel electrophoresis.
 　
         
 
 
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