Proteins HPLC analysis

The second example is the SE-HPLC analysis of recombinant hGH. In this example, SE-HPLC is used for both a purity and a protein concentration method for bulk and formulated finished products. This method selectively separates both low molecular weight excipient materials and high molecular weight dimer and aggregate forms of hGH from monomeric hGH, as shown... 

The purified protein was subjected to reversed-phase HPLC analysis by using a 150 X 1 mm Cig column with gradient elution from 0.1% aqueous trifluo-roacetic acid (TEA) to 0.1% TEA in acetonitrile, over a period of 55 min, at a flow... 

DeMar Jr. J.C., Disher, R.M., and Wensel, T.G. (1992) HPLC analysis of protein-linked fatty acids using fluorescence detection of 4-(diazomethyl)-7-diethylaminocoumarin derivatives Abstract 465. Biophys. J. 61(A81). 

A semipermeable membrane is placed between two liquids, and the analytes transfer from one liquid to the other. This technique is used for investigating extracellular chemical events as well as for removing large proteins from biological samples prior to HPLC analysis. 

Most HPLC instruments monitor sample elution via ultraviolet (UV) light absorption, so the technique is most useful for molecules that absorb UV. Pure amino acids generally do not absorb UV therefore, they normally must be chemically derivatized (structurally altered) before HPLC analysis is possible. The need to derivatize increases the complexity of the methods. Examples of derivatizing agents include o-phthaldehyde, dansyl chloride, and phenylisothiocyanate. Peptides, proteins, amino acids cleaved from polypeptide chains, nucleotides, and nucleic acid fragments all absorb UV, so derivatization is not required for these molecules. 

The DCL was first composed in the absence of the CA template, a so-called blank DCL. Equilibration was complete in 24 hours, with each of the expected imine reduction products being observed in the HPLC trace (Fig. 2.2). Reconstitution of the DCL in the presence of a stoichiometric quantity of enzyme afforded the second trace. Equilibration was significantly retarded in the presence of the protein, 2 weeks being necessary for a dynamic equilibrium to be realized. A thermal denaturation step preceded HPLC analysis. 

HPLC analysis, aided by the4-methylumbelliferyl chromophore appended to the carbohydrate anomeric position, identified all 12 of the expected amines. Equilibration in the presence of a stoichiometric quantity of HEWL led to a small amplification of two components 22b and 22f, which could be augmented by using an excess of protein as the DCL template (Fig. 2.6). As a control, the DCL was constructed in the presence of chitotriose, a good HEWL inhibitor, and no amplification could be observed. 

The DCL was created under standard disulfide exchange conditions at pH 7.5. After equilibration for 48 hours, each of the expected 15 disulfide products could be observed in the library using LC-MS analysis (Fig. 2.11). The library was then equilibrated in the presence of CaM, followed by a centrifugation filtration step to separate protein/bound components from free components in solution. Analysis of the filtrate was complicated by the filtration membrane affecting the composition of the library. The bound components, however, provided meaningful results. Denaturation and filtration afforded a mixture of all peptides that had bound to CaM in the course of the DCL. HPLC analysis indicated significant amplification of dimer cc (80%) and a small amplification of dimer ec (10%). Resynthesis of these two components and binding assay established values of 10 and... 

HPLC analysis of food proteins and peptides can be performed for different purposes to characterize food, to detect frauds, to assess the severity of thermal treatments, etc. To detect and/or quantify protein and peptide components in foods, a number of different analytical techniques (chromatography, electrophoresis, mass spectrometry, immunology) have been used, either alone or in combination. The main advantages of HPLC analysis lie in its high resolution power and versatility. In a single chromatographic run, it is possible to obtain both the composition and the amount of the protein fraction and analysis can be automated. 

Meat proteins comprise a water-soluble fraction (containing the muscle pigment myoglobin and enzymes), a salt-soluble fraction composed mainly of contractile proteins, and an insoluble fraction comprising connective tissue proteins and membrane proteins. As reviewed by Dierckx and Huyghebaert [107], HPLC analysis of meat proteins has been successfully applied to evaluate heat-induced changes in the protein prohle, to detect adulterations (addition of protein of lower value, the replacement of meat from high-value species with meat from lower-value species, etc.), and for specie identification in noncooked products (also for fish sample). 

Figure 17 Elution Profiles for the Analytical RP-HPLC Analysis for the Purified (Insert Panel) and the Preparative RPC of the Crude Synthetic 22-mer Polypeptide Derived from the N-Terminal Secretory Leader Sequence of the Sperm Tail Protein, tpx-1, a Member of the Cystine-Rich Secretory Protein Superfamily 161 ab...

Cheever, K.L., Richards, D.E. Plotnick, H.B. (1980) Metabohsm of ortho-, meta-, and para-toluidine in the adnlt male rat. Toxicol, appl. Pharmacol, 57, 361-369 Cheever, K.L., DeBord D.G, Sweaiengin, T.F. Booth-Jones, A.D. (1992) ortho-Toluidine blood protein addnets HPLC analysis with fluoreseenee deteetion after a single dose in the adult male rat. Fundam. appl Toxicol, 18, 522-531 Chemfinder (2000) ortiho-Toluidine Hydrochloride [http //www.ehemfinder.com/result.asp] Chemieal Information Serviees (1999) Directory of World Chemical Producers (Version 99.1.0) [CD-ROM], Dallas, TX... 

When Fab-B(SH) has been eluted, connect a second, larger Sephadex G25 column (2 6-cm diameter, bed height 20 cm) to the chart recorder. After the 30 min-mcubation, load the Fab-A(SH)/o-PDM/DMF mixture onto this second G25 column, and elute at a flow rate of approx 200 mL/h. Collect the Fab-A(mal) protein peak (elutes after 8-10 min), taking a 45-pL sample for HPLC analysis Stop collecting when the chart recorder pen has returned half way to baseline to avoid contamination with o-PDM/DMF, which elutes as a large second peak... 

B 2 The new reagent 4-Ar,A7-dimethyIaminoazobenzene-4 -isothiocyanate (DABITC) is often used for the Edman method of protein sequence analysis Wnte a reaction showing this use of the reagent 3 Describe how the HPLC and CE instruments detected the FMOC-amino acids eluting from the column... 

Compared to refined vegetable oils, the compositions of crude vegetable oils and oil and fat products are more complicated. These samples contain proteins, carbohydrates, and minerals that interfere with HPLC separation and reduce the lifetime of the HPLC column. These compounds need to be largely eliminated from the extract before HPLC analysis. Saponification and heating are used to weaken sample matrices to allow the solvent to fully access all tocopherols and tocotrienols of the sample. Liquid/liquid extraction is used to remove these polar compounds from the organic solvent layer that contains tocopherols and tocotrienols. The normal-phase HPLC method is usually used for crude vegetable oils and vegetable oil products reversed-phase HPLC can be used for animal fat products. 

The crude extract needs to be further purified for HPLC analysis. Direct injection of the crude extract into the HPLC would clog the frit and analytical column with precipitated impurities (i.e., proteins). 

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