known to be absent, the use of either 0.2 or 1% PTA would be satisfactory. Wang and Smith (5) and Cad- man et al. (6) reported that protein treated with deter- gent often caused a precipitate to form when the Folin reagent was added during the Lowry assay. Purifica- tion of the protein with TCA and PTA in the present study did not prevent this. Hence, the practice (5, 6) of adding SDS prior to the Folin reagent was adopted when detergents were present in the test sample. Ad- dition of SDS in this step dilutes the reaction mixture and reduces test sensitivity slightly. Absorbance read- ings are higher where this step can be omitted, either where detergent is absent or where the detergent present is SDS itself (compare absorbance values in Figs. 1 and 3 with corresponding values in Figs. 2 and 4 where this step was omitted). The tolerance limits for detergents (1% for SDS and 0.1% for Tween 20, Triton X-100, and Nonidet P-40) in protein samples apply specifically to the microassay (4). This assay has been modified for high sensitivity by increasing the proportion of the test sample in the Lowry reaction mixture. Thus, the test sample makes up 57% of the reaction mixture volume (prior to adding the Folin reagent). Higher detergent concentration can be tolerated in a typical “standard” Lowry assay where the test sample might be no more than about 5–10% of the total volume. Conclusion In purifying proteins by acid precipitation, 5% TCA precipitates proteins (exemplified by ovalbumin and latex proteins) poorly in the presence of SDS. However, the inhibitory effect of SDS on protein precipitation is effectively overcome by a combination of 5% TCA and 1% PTA. The TCA/PTA mixture also precipitates pro- teins readily in the presence of 0.1% Tween 20, Triton X-100, or Nonidet P-40. TCA/PTA has the capacity to precipitate a wider cross-section of proteins than TCA alone, the combination of acids remaining effective in the presence of SDS and the other detergents exam- ined in this study. REFERENCES 1. Lowry, O. H., Rosebrough, N. R., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265–275. 2. Peterson, G. L. (1979) Anal. Biochem. 100, 201–220. 3. Yusof, F., and Yeang, H. Y. (1992) J. Natl. Rubber Res. 7, 206 – 218 4. Yeang, H. Y., Yusof, F., and Abdullah, L. (1995) Anal. Biochem. 226, 35– 43. 5. Wang, C. S., and Smith, R. L. (1975) Anal. Biochem. 63, 414 – 417. 6. Cadman, E., Bostwick, J. R., and Eichberg, J. (1979) Anal. Bio- chem. 96, 21–23. 7. Sunderasan, E., and Yeang, H. Y. (1993) J. Natl. Rubber Res. 8, 293–298. 8. Tomazic, V. J., Withrow, T. J., Fisher, B. R., and Dillard, S. (1992) Clin. Immunol. Immunopathol. 64, 89 –97. 9. Turjanmaa, K., Alenius, H., Ma ¨ kinen-Kiljunen, S., Reunala, T., and Palosuo, T. (1996) Allergy 51, 593– 602. A Screening Approach for Selection of Clones Simultaneously Mutagenized at Multiple Sites Dimiter Demirov,* Alexey Savov,† Ivo Kremensky,† and Varban Ganev* *Department of Chemistry and Biochemistry and †Department of Molecular Pathology, Medical University of Sofia, Sofia 1431, Bulgaria Received June 2, 1998 Site-directed mutagenesis of DNA fragments is widely used for studying the structure and function of the encoded proteins. The most commonly used ap- proach is obtaining the target DNA fragment into a single-stranded form and extending the second DNA strand with a partially complementary mutagenic primer (1). Applying the mutagenesis on uridylated templates (2), the efficiency of mutagenesis with a sin- gle mutagenic primer may exceed 80%, which usually obviates the necessity of preliminary screening for mu- tagenized clones. Currently, most of the site-directed mutagenesis experiments aim at introducing muta- tions into the target DNA at multiple sites. This ap- proach requires repeated time-consuming steps: primer extension and ligation, transformation, se- quencing of several random clones to identify the de- sired mutant, and isolation of partially mutagenized DNA for the next round of mutagenesis. Moreover, each consecutive step of primer extension bears the risk of introducing uncontrolled random mutations, thus compromising the success of previous ones. Simul- taneous mutagenesis with more than one mutagenic primer at multiple sites is supposed to get rid over these problems, but has not been used yet, because it could lead to a mixture of differently mutagenized clones at low abundancy (3) and the identification of the desired clone(s) would require extensive sequenc- ing work. Therefore, an appropriate screening proce- dure for selection of mutagenized clones would make simultaneous mutagenesis a more attractive procedure in experiments aiming at multiple sites mutagenesis at increased throughput. Here, a single-strand conformation analysis (SSCA) 1 screening approach for identification of low-abundance clones simultaneously mutagenized at multiple sites 1 Abbreviation used: SSCA, single-strand conformation analysis. 384 NOTES & TIPS ANALYTICAL BIOCHEMISTRY 265, 384 –386 (1998) ARTICLE NO. AB982881 0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.