By Charles A. Lucy, Nicole E. Baryla (auth.), Mark A. Strege, Avinash L. Lagu (eds.)
Capillary electrophoresis (CE) is without doubt one of the optimal tools for the fast research of pharmaceutical proteins and peptides, and hence now much more very important with the sequencing of the human genome and the next creation of proteomics. In Capillary Electrophoresis of Proteins and Peptides, best educational scientists and specialist laboratorians operating within the pharmaceutical describe their top CE strategies in step by step element for simple copy. The authors current state-of-the-art thoughts for either capillary coatings and analytical detection through laser-induced fluorescence, for the advance and commercialization of biopharmaceuticals, and for affinity capillary electrophoresis within the assessment of protein binding, together with using protein cost ladders. extra chapters talk about CE and capillary isoelectric focusing, mixed with electrospray mass spectrometry detection, for appearing proteomic reviews. components of specified curiosity coated contain proteomic functions, in addition to purposes within the box of protein-ligand binding. The protocols stick with the profitable tools in Molecular Biology™ sequence layout, every one supplying step by step laboratory directions, an creation outlining the main in the back of the strategy, lists of kit and reagents, and tips about troubleshooting and fending off recognized pitfalls.
accomplished and cutting-edge, Capillary Electrophoresis of Proteins and Peptides will end up necessary to either new and skilled investigators conducting major protein and peptide experiences in biotechnology, drug discovery, and pharmaceutical study and development.
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Additional info for Capillary Electrophoresis of Proteins and Peptides
Detection limits for CE-LIF have been reported from attomole to zeptomole and even to single molecules (4,5). The work of an ultraviolet light-emitting diode (LED)-induced fluorescence was first carried out by Hillebrand et al. (6), who separated and detected within femtomole range a mixture of amino acids labeled with fluorescamine. A UVLED with emission maximum at 370 nm (∆λ = 12 nm) was used as an excitation source for fluorescamine-derivatized analytes. A scheme of the experimental setup is shown in Fig.
One of the problems frequently experienced when using visible LIF detection is matrix autofluorescence which has the effect of degrading the overall sensitivity of the technique. However, the use of near-infrared (NIR) laser induced fluorescence nearly eliminates matrix autofluorescence, as very few molecules have intrinsic fluorescence in this region. This chapter describes the use of covalent and noncovalent labeling schemes for tagging biomolecules with near infrared dyes. To fully appreciate the advantages that the NIR LIF technique can supply, we also review applications that employ detection schemes other than NIR LIF.
The Grignard-based sublayer has been employed further to compare the performance of all four monomers. The procedures proved to be very practical for reducing protein adsorption on capillary walls in CE. As aforementioned, the coating of capillaries can be based either on silanization or on the Grignard reaction (see Figs. 3 and 4). 1 M NaOH is flushed through the capillary (4–5 m long, 50 µm id) for 1 h followed by flushing with water for another hour. Finally, a 4% solution of 3-methacryloxypropyltrimethoxysilane in a 1:1 mixture of glacial acetic acid and water should be prepared, and the capillary has to be flushed with this solution for 20 min.