Figure 2.

Purification and identification of allele-specific proteins bound to the region containing SNP rs163184. (A) Oligos immobilized on the nanobeads. Oligos 6 and 7 were designed for the risk and the NR alleles of SNP rs2074196, respectively, as described in our previous study (16), and were included to demonstrate the reproducibility of the purification system using nuclear extracts from INS-1 cells. Oligo 5 was designed against the predicted PPARA binding site, which was also included in oligos 3 and 4. However, further analysis identified no binding between PPARA and this region. (B) Aliquots of purified fractions were separated using SDS-PAGE (5–20% gradient gel) and visualized using silver staining. (C) The same samples (panel B, lanes 3 and 4) were separated using longer SDS-PAGE (5–20% gradient gel) and visualized using silver staining to allow for the excision of the specific protein bands. The names and locations on the gel of polypeptides that were analyzed using ion-spray mass spectrometry are indicated by corresponding numbers. Polypeptide band 4 was broad and part of the band was also present in the oligo 4 (risk) lane, designated as polypeptide band 4′ and used in further analysis. SNP, single nucleotide polymorphism; oligo, oligonucleotides; NR, non-risk.