Study of protein–protein binding reaction by whole‐column fluorescence‐imaged CIEF

Abstract

Whole-column fluorescence-imaged CIEF was applied to study protein-protein binding reaction. A homemade whole-column fluorescence-imaged CIEF experimental setup was built, and its CIEF performance was evaluated with native fluorescent protein green fluorescence protein and fluorescently labeled proteins (bovine albumin, human albumin, and BSA). pIs, focusing time, detection limits, and linear quantitative range of the proteins were obtained. Furthermore, the method was used to study FITC-protein A-human IgG binding reaction. Experimental results showed that the apparent binding ratio of the FITC-protein A to human IgG was 1:2, and pI of the binding conjugates were about 6.3-6.5. No binding reaction was found between green fluorescence protein and the fluorescent-labeled proteins.

Recent developments in CE and CEC of peptides (2009–2011)

“…nanoliter detection cell volume with millisecond time resolution. The earlier developed special detection cell constructions with increased optical path and thus enhanced sensitivity (Z‐shaped cell, bubble cell or sleeve cell), and some new designs of the UV‐absorption detector for capillary and chip separations are described in the review on optical detection schemes 161.…”

“…Lamp (Xenon‐Mercury)‐based fluorescence and especially laser‐induced fluorescence (LIF) represent the most sensitive detection schemes in CE, CEC and other microanalytical systems as evidenced in the reviews on advances in optical detection methods generally 161 and fluorescence detection particularly 166, 167. With special designs of detection cells, such as liquid sheath‐flow cuvettes and integrated separation and detection devices, LIF detection is able to detect proteins and peptides at femtomolar concentration LODs and zeptomole substance amount LODs 168 and has a potential to achieve the detection limit of few or even of a single molecule 169, 170.…”

Fully integrated PDMS/SU‐8/quartz microfluidic chip with a novel macroporous poly dimethylsiloxane (PDMS) membrane for isoelectric focusing of proteins using whole‐channel imaging detection

“…Several commercial CIEF instruments have been developed and are available in the market as powerful separation systems. For example, the iCE280 analyzer invented by Convergent Bioscience is such an instrument utilizing CIEF for the separation of proteins and biomarkers 4–6. This instrument has been widely adopted by the pharmaceutical industry.…”