Protein Protocols Protein Protocols

Phosphopeptide Mapping 679
until the solvent line diffuses to the dye position at the top of the TLC plate, ~3 cm below
the top of the plate. This usually takes 7–8 h (see Notes 4 and 5).
7. Remove the TLC plate from the chamber and dry for >1 h. Expose the TLC plate to ARG
film or a phosphor-imager screen overnight, and develop. If the signal is too weak, expose
for 5–7 d. The use of a phosphor-imager greatly diminishes the length of time required to
obtain a 2-D phosphopeptide map (see Fig. 3).
4. Notes
1. We routinely label ~1 × 106 cells; however, depending on the abundance of the specific
protein of interest and on the number of phosphorylation sites, this number may vary from
1 × 105 to 1 × 107 cells. In addition, adherent and nonadherent cells may both be labeled in
suspension. The cells can be labeled with 32P-orthophosphate in a 15- or 50-mL disposable
conical tube or T-flask. Please note that the activity of kinase(s)/phosphatase(s)
present in adherent cells may be altered when labeling in suspension. Dish size is not
important as long as enough media are added to cover the bottom of the dish/flask. Attempt
to achieve a final 32P-orthophosphate concentration of ~1.0–1.3 mCi/mL of media.
2. We use ELB to lyse cells and generate cellular extracts; however, any extraction buffer
containing Triton X-100, SDS, DOC, NP40, or similar detergent that will lyse cells will
suffice. If a strong background is observed following the SDS-PAGE, transferring the
immobilized protein A agarose immunocomplexes to a new microcentrifuge tube prior to
the final wash and centrifugation step can result in a reduced background with minimal
loss of specific signal.
3. When running multiple lanes of the same 32P-labeled protein immunocomplexes on SDS-
PAGE, treat each lane/NC filter slice separately. The trypsinized peptides from as many
as five NC filter slices can be combined together, then frozen on dry ice, and dried.
4. Seal the top of the TLC chamber with vacuum grease, and minimize the amount of time
the lid is off of the chamber. Pre-equilibrate chamber for >48 h prior to use. We routinely
change the TLC chamber buffer every 8 wk. Poor separation in the second dimension is
usually indicative of buffer alterations owing to evaporation and/or hydration.
5. The procedures described in this chapter can be stopped at the following steps:
a. When the immunocomplexes are in 2X sample buffer following the immunoprecipitation.
b. After lyophilization following trypsinization of the NC filter slices.
c. After drying the TLC plate following the first-dimension electrophoresis.
Acknowledgments
We thank Fung Chen for protocols and Jeff Settleman for the “1g spin.” S. F. D. is an
Assistant Investigator of the Howard Hughes Medical Institute.
References
1. Kamp, M. P. and Sefton, B. M. (1989) Acid and base hydrolysis of phosphoproteins bound
to immmobilon facilitates analysis of phosphoamino acid in gel-fractionated proteins.
Analyt. Biochem. 176, 22–27.
2. Lees, J. A., Buchkovich, K. J., Marshak, D. R., Anderson, C. W., and Harlow, E. (1991)
The retinoblastoma protein is phosphorylated on multiple sites by human cdc2. EMBO 13,
4279–4290.
3. Luo, K., Hurley, T. R, and Sefton, B. M. (1991) Cynogen bromide cleage and proteolytic
peptide mapping of proteins immobilized to membranes. Meth. Enzymol. 201, 149–152.
4. Desai, D., Gu, Y., and Morgan, D. O. (1992) Activation of human cyclin-dependent kinase
in vitro. Mol. Biol. Cell. 3, 571–582.