Performance of agarose IEF gels as the first dimension support for non-denaturing micro-2-DE in the separation of high-molecular-mass plasma proteins and protein complexes

Agarose micro-column gels (1% w/v agarose, diameter 1.4 mm and length 35 mm) were prepared as the first-dimension IEF support for non-denaturing 2-DE and the performance was compared with that of polyacrylamide gels (4.2% T and 4.8% C, the same gel size) using a human plasma sample. Sorbitol was not added in the agarose IEF gels, since its presence not only delayed the focusing of the proteins but also deteriorated the protein resolution. The optimum IEF time of the agarose gels for separation of 2 mu L plasma sample (ca. 120 mu g proteins) was decided to be 46 min, which is much shorter than that of the polyacrylamide gels (75 min). MALDI-MS and PMF assignment of the spots on the micro-2-DE gels at apparent molecular mass above ca. 5 x 10(2) kDa and pI from 4 to 8 revealed that when polyacrylamide IEF gels were used, many of the high-molecular-mass proteins resided at the sample loading edge or in basic pI regions as smear bands. When agarose IEF gels were used, most of the high-molecular-mass proteins moved to more acidic pI positions and were better focused, and their apparent p I values matched well with those previously reported for purified proteins. These results demonstrated the advantages of agarose-IEF/2-DE for the separation of high-molecular-mass proteins and protein complexes under non-denaturing conditions.