Immunoscreening of Fusion Proteins Produced in Lambda Plaques

Thomas P. St. John1

1 Fred Hutchinson Cancer Research Center, Seattle, Washington
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 6.7
DOI:  10.1002/0471142727.mb0607s09
Online Posting Date:  May, 2001
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Abstract

Screening large numbers of plaques containing particular proteins is accomplished by techniques that are analogous to those described for screening with radioactive DNA probes. However, in the basic protocol described here, the plaques are screened with antibodies specific to the desired proteins. An alternate protocol provides a method for increasing the amount of recombinant protein in each plaque by inducing expression from the lac promoter that directs its expression.

     
 
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Table of Contents

  • Section IV: Screening with Antibodies
  • Immunoscreening of Fusion Proteins Produced in Lambda Plaques
  • Basic Protocol 1: Screening a λgt11 Expression Library with Antibodies
  • Alternate Protocol 1: Induction of Fusion Protein Expression with IPTG Prior to Screening with Antibodies
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Screening a λgt11 Expression Library with Antibodies

  Materials
  • λgt11 cDNA expression library
  • 150‐mm LB plates (unit 1.1)
  • E. coli LE392 (Table 97.80.4711)
  • 1% LB top agar (unit 1.1)
  • 0.05% (v/v) NaN 3 in India ink (optional)
  • recipeImmunoscreening buffer
  • First‐stage antibody
  • 125I‐labeled second‐stage reagent reactive with first‐stage antibody
  • 132‐mm nitrocellulose membrane filters
  • Additional reagents and equipment for titering and plating bacteriophage (units 1.11 & 6.1) and 3.NaNautoradiography ( 3.NaN)
NOTE: All materials coming into contact with E. coli must be sterile.

Alternate Protocol 1: Induction of Fusion Protein Expression with IPTG Prior to Screening with Antibodies

  Additional Materials
  • E. coli Y1090 (Table 97.80.4711)
  • 10 mM IPTG (Table 97.80.4711)
  • 42°C room or incubator
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Figures

Videos

Literature Cited

Literature Cited
   Erlich, H.A., Cohen, S.N. and McDevitt, H.O. 1978. A sensitive radioimmunoassay for detecting products translated from cloned DNA fragments. Cell 13:681‐689.
   Helfman, D.M., Feramisco, J.R., Fiddes, J.C., Thomas, G.P., and Hughes, S.H. 1983. Identification of clones that encode chicken tropomyosin by direct immunological screening of a cDNA expression library. Proc. Natl. Acad. Sci. U.S.A. 80(1):31‐35.
   Kemp, D.J. and Cowman, A.F. 1981. Direct immunoassay for detecting Escherichia coli colonies that contain polypeptides encoded by cloned DNA segments. Proc. Natl. Acad. Sci. U.S.A. 78(7):4520‐4524.
   Skalka, A. and Shapiro, L. 1976. In situ immunoassays for gene translation products in phage plaques and bacterial colonies. Gene 1:65‐79.
   Young, R.A. and Davis, R.W. 1983. Efficient isolation of genes by using antibody probes. Proc. Natl. Acad. Sci. U.S.A. 80(5):1194‐1198.
Key Reference
   Huynh, T.V., Young, R.A. and Davis, R.W. 1984. Construction and screening cDNA libraries in λgt10 and λgt11. In DNA Cloning: A Practical Approach, Vol. 1, (D.M. Glover, (ed.) pp. 49‐78. IRL Press, Oxford.
  Provides an excellent description of immunological screening procedures.
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