Immunoscreening after Hybrid Selection and Translation

Baruch Velan1

1 Israel Institute for Biological Research, Ness Ziona, Israel
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 6.8
DOI:  10.1002/0471142727.mb0608s13
Online Posting Date:  May, 2001
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Abstract

In this procedure, plasmid cDNA clones are screened for their ability to select a particular mRNA. Plasmid DNA is bound to nitrocellulose filters, hybridized to mRNA, washed, and the selected mRNA is eluted from the filter. Eluted mRNA is characterized by translation into 35Sā€labeled protein, which is identified by immunoprecipitation and denaturing (SDS) polyacrylamide gel electrophoresis. The desired clone is that which is able to select an mRNA that translates into the desired protein. This procedure can be modified to characterize cosmids and bacteriophage DNA.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1:

  Materials
  • Brain‐heart‐infusion (BHI) medium (37.5 g/liter, autoclaved) containing appropriate antibiotics
  • Chloramphenicol (Table 97.80.4711)
  • TE buffer, pH 7.6 ( appendix 22)
  • 1 M NaOH
  • recipeNeutralization solution
  • 6× SSC ( appendix 22)
  • recipeHybridization solution IV
  • Poly(A)+ mRNA (unit 4.5)
  • recipe65°C TES buffer in 0.5% SDS
  • recipe65°C TES buffer
  • 10 mg/ml yeast tRNA
  • Buffered phenol (unit 2.1)
  • 50:1 chloroform/isoamyl alcohol
  • 3 M sodium acetate, pH 5.2
  • Ethanol
  • recipeTranslation mixture
  • [35S]methionine (800 Ci/mmol)
  • recipeImmunoprecipitation buffer
  • Nonimmune serum
  • Protein A–Sepharose suspension
  • Polyclonal or monoclonal antibodies ( Chapter 11)
  • recipeHigh‐salt immunoprecipitation buffer
  • 2× SDS/sample buffer (unit 10.2)
  • 96‐well microtitration dish
  • Beckman JS‐4.2 rotor or equivalent
  • Sterile 15‐ml capped glass culture tubes
  • 0.45‐µm nitrocellulose filters (2.5‐cm diameter)
  • Multifilter washing apparatus
  • 80°C vacuum oven
  • Sterile 1.8‐ml round‐bottom plastic tubes (Nunc)
  • Sterile silanized 1.5‐ml microcentrifuge tubes ( appendix 3A)
  • Sterile needles
  • Additional reagents and equipment for preparation of plasmid DNA (unit 1.6) and denaturing (SDS) polyacrylamide gel electrophoresis (unit 10.2)
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Figures

Videos

Literature Cited

Literature Cited
   Harpold, M.M., Dobner, P.R., Evans, R.M., and Bancroft, F.C. 1978. Construction and identification by positive hybridization translation of a bacterial plasmid containing a rat hormone structural gene sequence. Nucl. Acids Res. 5:2039.
   Lemke, G. and Axel, R. 1985. Isolation and sequence of a cDNA encoding the major structural protein of peripheral myelin. Cell 40:501.
   March, C.J., Mosley, B., Larsen, A., Cerret, D.P., Braedt, G., Price, V., Gillis, S., Henney, C.S., Krunheim, S.P., Grabstein, K., Canlon, P.J., Hopp, P., and Cosman, D. 1985. Cloning, sequence, and expression of two distinct human Interleukin I cDNAs. Nature 315:641.
   Parnes, J.R., Velan, B., Felsenfeld, A., Ramanathan, L., Ferrini, U., Appella, E., and Seidman, J.G. 1981. Mouse β2‐microglobulin cDNA clones: A screening procedure for cDNA clones corresponding to rare mRNAs. Proc. Natl. Acad. Sci. U.S.A. 78:2253.
   Ricciardi, R.P., Miller, J.S., and Roberts, B.E. 1979. Purification and mapping of specific mRNAs by hybridization selection and all free translation. Proc. Natl. Adac. Sci. U.S.A. 76:4921.
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