Detection of binding artifacts in gel electrofocusing using a polymeric dye probe

Poly R‐480, an amphoteric dye of molecular weight 160 000,is the product of copolymerization reaction and can be expected to yield a polydisperse pattern in polyacrylamide gel isoelectric focusing (PAGIF). Such is indeed obtained in conventional PAGIF using commercial synthetic carrier ampholyte mixtures but not in buffer PAGIF, in which a few discrete dye bands are produced. Upon excision of single bands and segments of zones in buffer PAGIF and PAGIF in SCAMs, respectively, the entire original pattern is reestablished in each case, thereby confirming the artifactual nature of both the simple band pattern as well as the polydisperse patten. The simple pattern in buffer PAGIF prevails in the presence of 8 M urea and 8 mM CHAPS, while the polydisperse pattern obtained in SCAMs is resolved into multiple bands in the presence of either one or both of these agents. These observations suggest: a) hydrophobic and hydrogen bond dye‐dye and/or dye‐SCAM interactions produce the artifactual polydisperse pattern in PAGIF in SCAMs, b) electrostatic dye‐dye interactions are responsible for the artifactual simple band pattern in buffer EF, and c) dye‐SCAM electrostatic interactions produce the artifactual multiple band pattern in the SCAM‐urea‐CHAPS system. These data clearly underline the need for critical experimentation in the evaluation of PAGIF bands and band numbers.