Methods Materials. Chemicals were purchased from Fisher Scientific, Pittsburgh, PA. All chemicals were of analytical grade or better. Corn on the cob with husk was purchase from Walmart. Some of the microorganisms were purchased from ATCC (Manassas, Virginia). Growth of microorganisms. The various organisms were grown until late log to early stationary phase (OD 0.9-1.3) under the following conditions. Thermus thermophilus (HB8, ATCC 27634) was grown overnight on Luria broth/agar plates. Individual colonies were grown overnight in Luria broth at 60 ºC to an OD of ~1.0. The archaea Sulfolobus acidocaldarius (ATCC 33090) was grown in #1723 ATCC medium (Revised Sulfolobus Medium, pH 3.0) at 70 ºC until the OD reached ~1.0. Thermus aquaticus (ATCC 25104) was grown in #461 ATCC medium (Castenholz TYE Medium, pH 7.6) at 70 ºC until the OD reached ~1.0. E. coli cells (BL21 DE3 strain, Invitrogen, Carlsbad, CA) were grown overnight on Luria broth/agar plates. Individual colonies were selected and grown overnight in Luria broth at 37 ºC until the OD reached ~0.9. Vibrio cholerae (O395N1) was grown overnight in ~15 ml Luria broth buffer and then transferred to 50 ml Luria broth buffer until the OD reached ~1.3. Bacillus subtilis (PE239) was grown at 30 ºC for 24 h in ~15 ml Luria broth buffer and then transferred to 50 ml Luria broth buffer until the OD reached ~1.2. Saccharomyces cerevisiae (Invitrogen, Carlsbad, CA) was grown overnight on Luria broth/agar plates. Individual colonies were grown overnight in Luria broth at 23 ºC until the OD reached ~1.2. Tetrahymena thermophila (WH-6, ATCC 30007) was grown in ATCC medium 357 upright at 25 ºC for 48-72 h. All eight microorganisms were harvested by centrifuging at 5500 rcf for 10 min and then resuspending the pellet in 25 mM potassium phosphate buffer (pH 6.8). Sodium chloride was added to a final concentration of 450 mM and the cells were lysed by sonicating for 10 min. Maize lysate preparation. The maize cell lysate was prepared from a fresh cob of corn with husk purchased at Walmart. The kernels were removed from the cob, and then washed with distilled water, drained, and incubated in 100 mM sodium chloride / 25 mM sodium phosphate buffer (pH 6.8). The mixed solution was grinded by using a 100w blender, and then lysed by sonication for 10 min. The cell debris was removed by centrifugation. Determination of lysate protein concentration. The cell lysates of all organisms were concentrated using a Nanosep Centrifugal Device (Pall, Port Washington, NY) with a 3kDa membrane. The total protein concentration in the lysates was estimated by measuring the absorbance at 280 nm using a Thermo a nanodrop nd1000 instrument (Wilmington, DE). The estimated final loading amount was ~300 µg protein per gel. Since the variable presence of nucleic acids in different organisms likely interferes with the calculation of protein concentration, the final loading amount has some variation. D2D SDS-PAGE analysis. The clear cell lysate of all organisms were analyzed by D2D SDS-PAGE as described previously (1). A description of D2D SDS-PAGE is found in Fig 1. All gels were stained with Commassie blue R250 (Fisher, Pittsburgh, PA). Gel pictures were taken using a Gel Doc Imager (Bio-rad, Hercules, CA).
References 1.
Xia, K., Manning, M., Hesham, H., Lin, Q., Bystroff, C., and Colon, W. (2007) Identifying the subproteome of kinetically stable proteins via diagonal 2D SDS/PAGE, Proc. Nat. Acad. Sci. USA 104, 17329-17334.
