Protein liquid chromatography, methods

Sheehan, D. (1996) Fast protein liquid chromatography, Methods Mol. Biol. 59, 269-275. 

C17. Crow, J. P., Measurement and significance of free and protein-bound 3-nitrotyrosine, 3-chlorotyrosine, and free 3-nitro-4-hydroxyphenylacetic acid in biologic samples A high-performance liquid chromatography method using electrochemical detection. Meth. Enzymol. 301, 151-160(1999). 

Pinkerton TC, Koeplinger KA (1990) Determination of warfarin-human serum albumin protein binding parameters by an improved Hummel-Dreyer high performance liquid chromatography method using internal surface reversed-phase columns. Anal Chem 62 2114-2122. 

Most of the procedures published so far have been either unsuitable for larger scale preparations (maximally 1 mg) or irreproducible. In an attempt to overcome these problems a purification procedure has been developed recently [108] which is a modification of a previous method [83]. The modified procedure involves FPLC (fast protein liquid chromatography), is rapid, reproducible and allows the preparation of large amounts of active enzyme. Purified transhydrogenase obtained by this procedure is reconstitutively active and pumps protons as judged by quenching of... 

Another example of on-line monitoring of enzyme activities was given by Kunnecke et al. [88], when a FIA-system was used for the determination of enzyme activities during protein purification by fast protein liquid chromatography (FPLC). Photometric assays for four different oxidases were established in a FIA-system extending the linear range by the so-called zone sampling method. The FIA-device was coupled to the FPLC unit behind a... 

Method 2 Fast Protein Liquid Chromatography (FPLC)... 

The following sections will describe some of the various methods of liquid chromatography suitable for separation and analysis of biological (macro)molecules. Such systems often use high pressures and rapid flow rates, and are sometimes loosely described as HPLC (high performance liquid chromatography) or FPLC (fast protein liquid chromatography). 

The initial PIA purification method was developed by Mack et al. (3). These authors used a different, two-step chromatography protocol involving size-exclusion and ion exchange chromatography on Sephadex G-200, Q-Sepharose, and S-Sepharose. A similar purification method has been described recently to isolate a PIA-related polysaccharide polymer in E. coli (7). Briefly, E. coli cells were incubated in 50 raM Tris-HCL buffer (pH 8.0), 100 mg lysozyme, and 0.1 M EDTA at room temperature for 2 h. Phenol/chloroform extraction steps were performed to separate protein and debris contamination from the polysaccharide. Samples were concentrated by ultrafiltration devices (10,000 MW cut off) and fractionated on a fast protein liquid chromatography (FPLC) system with a Sephacryl S-2000 column (equilibration and elution buffer 0.1 MPBS, pH 7.4). 

One area that looks distinctly promising is the further application of immunochemical methods in fast protein liquid chromatography. The ingenuity already shown in the development of immunochemical assay procedures, not just with proteins but more generally, suggests at least one likely growth area for a more widespread application in f>ost-chronuitographic derivatization. 

Turanek J, Zaluska D, Neca J. Link of a fast protein liquid chromatography system with a stirred thermostated cell for sterile preparation of liposomes by the proliposome-Uposome method appUcation to encapsulation of antibiotics, synthetic peptide immu-nomodulators, and a photosensitizer. Anal Biochem 1997 249 131-9. 

See also-. Electrophoresis Isoelectric Focusing Polyacrylamide Gels Two-Dimensional Gels Proteins. Liquid Chromatography Liquid Chromatography-Mass Spectrometry Biotechnology Applications. Mass Spectrometry Ionization Methods Overview Electrospray Matrix-Assisted Laser Desorption/lonization Multidimensional Peptides and Proteins. Proteins Traditional Methods of Sequence Determination. 

---