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Muller, D. N. Wang, S. Denda, J. J. Meneses, R. A. Pedersen, and L. F. Reichardt, Cell 88, 603 (1997). 11 T. Takahashi, F. Nakamura, and S. M. Strittmatter. J. Neurosci. 17, 9183 (1997). 12 Y. Yang, G. Drossopoulou, P. T. Chuang, D. Duprez, E. Marti, D. Bumcrot, N. Vargesson, J. Clarke, L. Niswander, A. McMahon, and C. Tickle, Development 124, 4393 (1997). 13 J. G. Flanagan, Curr. Biol. 10, R52 (2000). 5 [2] AP FUSION PROTEINS AS in Situ PROBES 21 also describe in situ procedures in which these affinity probes are used to detect the distribution of cognate ligands or receptors in tissues or cells.

A. Kolodziej and R. A. Young, Methods Enzymol. 194, 508 (1991). TABLE I COMMONLY USED EPITOPE TAGS Tag Recognized sequence Advantages Disadvantages Original 12CA5 antibody not optimized for use in epitope tagging 9E10 monoclonal may not immunoprecipitate reliably. Endogenous c-myc expression interferes with use as epitope tag Detection by some antibodies requires placement at the protein termini Some commercially available antibodies require additional amino acids to specify recognition HA YPYDVPDYA Highly specific second-generation antibodies available Myc EQKLISEEDL FLAG DYKDDDK Polyhistidine HHHHHH Hybridoma line expressing the 9E10 monoclonal obtainable from the ATCC for use in large-scale projects Epitope easily cleaved off of tagged protein after purification Tagged proteins can be purified on Ni2ϩ affinity matrix.

S. Prickett, D. C. Amberg, and T. P. Hopp, BioTechniques 7, 580 (1989); (7) B. L. Brizzard, R. G. Chubet, and D. L. Vizard, BioTechniques 16, 730 (1994); (8) R. G. Chubet and B. L. Brizzard, BioTechniques 20, 136 (1996); (9) T. P. Hopp, K. S. Prickett, V. L. Price, R. T. Libby, C. J. March, D. P. Cerretti, D. L. Urdal, and P. J. Conlon, BioTechniques 6, 1204 (1988); (10) E. Hochuli, W. Bannwarth, H. Dobeli, and R. Gentz, Bio/Technology 6, 1321 (1988). TABLE II OTHER EPITOPE TAGS Tag Recognized sequence AU1 DTYRYI AU5 TDFYLK IRS RYIRS B-tag Universal QYPALT HTTPHH S-Tag Protein C KETAAAKFERQHMDS EDQVDPRLIDGK Glu-Glu KT3 VSV T7 HSV EYMPME or EFMPME PPEPET MNRLGK MASMTGGQQMG QPELAPEDPED a Comments Ref.

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