Electrophoretic profiling of both RNA and protein from a single 250-pL sample.
A novel approach is described that uses capillary electrophoresis (CE) to electrophoretically sample and separate both protein and RNA from a single injected plug of cell lysate. A 250-pL sample of lysate from Chinese hamster ovary cells (9.6 x 10(7) cells/mL) was hydrodynamically injected into a capillary containing a Tris-based aqueous buffer. This was followed by selective electrokinetic ejection of RNA from the lysate into water, yielding an effective cell concentration of RNA of 3000 cells/mL. The cellular components (e.g., proteins) retained in the capillary were separated and then detected with laser-induced fluorescence (LIF) using 275-nm excitation. The ejected/diluted sample was subsequently injected into a separate CE-LIF system, which utilized an entangled polymer sieving matrix and 543-nm excitation for the detection of ethidium bromide-labeled nucleic acids (i.e., RNA). Virtually no sample preparation is required other than simple washing and lysing of the cells isolated from culture. This combined approach can be easily modified for the detection of any analyte through adjustment of CE-HF conditions. In addition, it provides an effective method for desalting cellular RNA samples having complex matrixes, which results in improved RNA injection efficiency and a 7600-fold effective signal enhancement over total lysate injection.
Zabzdyr
Jennifer L
JL
Department of Chemistry, University of California, Riverside 92521, USA.
Lillard
Sheri J
SJ
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D019075:Electrophoresis, Capillary / Q000379:methods*; D011506:Proteins / Q000032:analysis*; D012313:RNA / Q000032:analysis*
D011506:Proteins; D012313:RNA