By Marie-Isabel Aguilar (auth.), Marie-Isabel Aguilar (eds.)
High functionality liquid chromatography (HPLC) performs a severe function at the present time in either our knowing of organic methods and within the improvement of peptide and protein-based prescription drugs. In HPLC of Peptides and Proteins: equipment and Protocols, top specialists from academia and comprehensively describe the way to effectively practice all of the severe HPLC thoughts wanted for the research of peptides and proteins. The tools variety from known strategies to these for capillary to large-scale preparative isolation. The authors have additionally awarded a couple of particular functions as case experiences to demonstrate the analytical techniques to a specific separation or assay problem, with examples drawn from modern fields in biochemistry and biotechnology. those functions comprise proteolytic mapping, posttranslational amendment, neuropeptide processing, glycopeptides and glycoproteins, MHC-binding peptides, toxins/venoms, membrane proteins, antibodies, combinatorial and proteome research, and enzymatic job. each one effortlessly reproducible protocol includes history notes, step by step directions, reagent and kit lists, and pointers on either troubleshooting and heading off recognized pitfalls.
entire and easy-to-follow, HPLC of Peptides and Proteins: tools and Protocols bargains each biologist and biotechnologist in any respect degrees of workmanship prepared entry to the robust instruments had to strengthen and perform peptide and protein separations and analyses today.
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Additional resources for HPLC of Peptides and Proteins: Methods and Protocols
Example text
Mono Q (HR5/5) prepacked column. Details specific for this column will be added to the method in italics. A linear gradient will be used to elute bound proteins, although conditions for the use of a step-elution system can readily be substituted for the gradient as will be indicated. An example chromatogram for this application is shown in Fig. 3. 1. Prepare the Gel/Column 1. Most IEX supports are available as preswollen gels or prepacked columns, but if working with one of the dry powder supports, it must first be preswollen at the pH to be used in the experiment according to the manufacturers instructions (normally 2–3 h at 90°C, or 48 h at room temperature) (see Note 4).
3. Halide-containing buffers will attack and corrode stainless steel high-pressure liquid chromatography (HPLC) systems, particularly frits, piston seals, and check valves. Leaching of metal ions can also interfere with some metal-sensitive proteins. Therefore, long-term use of these systems for ion-exchange chromatography is not recommended. If a stainless steel HPLC is used for IEX, wash the system with distilled or deionized H2O as soon as possible after completion of the experiment. 4. Do not use a magnetic stirrer with any ion-exchange supports, as this can break gel particles.
As IEX is an adsorptive technique, columns are normally short, with a bed height of 5–15 cm (1). Scaling-up of IEX columns is readily achieved by increasing the column diameter (1). 3. Method The following procedures describe ion-exchange chromatography on an anion-exchange support, using as an example the strong anion-exchanger 36 Stanton Fig. 3. Example of a typical ion-exchange column chromatogram. Initially, the ionexchange column is equilibrated in the starting buffer (low ionic strength) until a stable baseline of UV absorbance at 280 nm (filled line) is achieved.