Membranes isoelectric focusing

Another limitation of 2D gels is that membrane proteins are underrepresented. Because membrane proteins account for approximately 30% of total proteins (Wallin and Von Heijne, 1998), this is a serious problem for characterization of the proteome. The relative lack of membrane proteins resolvable on 2D gels can be attributed to thee main factors (i) they are not abundant, and therefore are difficult to detect by standard staining techniques, (ii) they often possess alkaline pi values, which make them difficult to resolve on the pH gradients most often used for isolelectric focusing, and (iii) the most important reason for under representation may be that membrane proteins are poorly soluble in the aqueous media used for isoelectric focusing (Santoni et al., 2000). Membrane proteins are designed to be soluble in lipid bilayers and are therefore difficult to solubilize in water-based solutions. 

Wang W, Guo T, Rudnick PA, et al. Membrane proteome analysis of micro-dissected ovarian tumor tissues using capillary isoelectric focusing/reversed-phase liquid chromatography-tandem MS. Anal. Chem. 2007 79 1002-1009. 

The specific labeled separated protein fractions blotted on a nitrocellulose membrane or specific immunoflxation-separated protein fractions in polyacrylamide after isoelectric focusing make it possible to detect some additional bands in CSF, i.e., IgM, IgA, free kappa or lambda light chains of specific antibodies (i.e., antiherpes, anti-borrelia, or anti-HIV) (LI, M3). 

Xanthine oxidase, which requires Fe, Mo and flavin adenine dinucleotide (FAD) as co-factors, is capable of oxidizing lipids via the production of superoxide radicals. It represents about 20% of the MFGM protein and part is readily lost from the membrane, e.g. on cooling isoelectric focusing... 

Rat brain microsome preparations were conveniently stored at -10C with retention of enzyme activity. Solubilization with Triton X-100 appears to be effective and the solubilized enzyme preparation, after filtration once with Amicon XM-300 diaflo membrane, was introduced into an isoelectric focusing column (LKB) with an ampholine pH range of 3.5-10. 

The basis for the multiplicity of the sialyltransferase activities remains to be elucidated. We plan to purify these enzyme species to homogeneity, using isoelectric focusing columns of smaller pH ranges in conjunction with affinity chromatography which has been successfully used to purify the soluble sialyl-transferases from bovine colostrum (57). Possibility exists that the heterogeneity of sialyltransferase activities as observed is due to differences in polypeptide sequences, carbohydrate content, or non-covalent interactions with other membrane components, and these possibilities can be clarified only with highly purified enzyme preparations. 

Capillary electrophoresis (CE) coupled to MS has the advantage of high resolution and soft ionization for biomolecules, which may be used to differentiate post-translational modifications and variants of intact proteins and oligonucleotides. Different modes of CE (capillary zone electrophoresis, capillary isoelectric focusing, capillary electrochromatography, micellar electrokinetic chromatography, nonaqueous capillary electrophoresis) to MS as well as online preconcentration techniques (transient capillary isotachophoresis, solid-phase extraction, membrane preconcentration) are used to compensate for the restricted detection sensitivity of the CE methodology [77, 78]. 

Masuoka, J., Glee, P.M., and Hazen, K.C. (1998). Preparative isoelectric focusing and preparative electrophoresis of hydrohobic Candida albicans cell wall proteins with in-line transfer to polyvinylidene difluoride membranes for sequencing. Electrophoresis 19, 675-678. 

In spite of the success and wide use, 2D gel is a relatively slow, labor-intensive and cumbersome technique, even with the availability of prefabricated strips for the first dimension (isoelectric focusing) and the pre-cast gels used in the second dimension. Another limitation is its inability to resolve lower abundance (less stable) proteins, membrane proteins, highly acidic or basic proteins, very large or small proteins, and hydrophobic proteins like G-coupled protein receptors with several transmembrane regions. 

Amino acid analysis is often touted as the most accurate method for determination of protein concentration. The data from this 1996 ABRF AAA study indicate that the vast majority of member facilities that participated in this study quantitate soluble protein well. The most striking aspect of this study, however, was the ability of the laboratories to identify the protein solely on its amino acid composition. The data from approximately 90% of the participants were sufficient for correct identification, if one knew the species of the protein s origin. Currently, identification of unknown proteins from AAA data is not frequently used for simple soluble proteins, such as triosephosphate isomerase. The technique is more commonly used to identify proteins that have been separated by two dimensional analysis on isoelectric focusing and SDS electrophoresis and then transferred to PVDF membranes. Such samples are usually present in low... 

Isotopic labelling Mass spectrometry Immunodetection Isoelectric focusing Glutathione labelling Specific quantitative Fast results, peptides sequencing, low amounts of sample Specific, detection in vivo Resolution Useful for in vivo studies, cross the plasmatic membrane Artefacts (in vivo) Qualitative, sample preparation, tedious results analysis No GSH antibodies available Sample preparation, slow, semiquantitative Qualitative and quantitative, optimization... 

Early experiments in the development of isoelectric focusing, a high-resolution steady-state electrophoresis method, occurred in 1912, with an electrolytic cell that was used to isolate glutamic acid from a mixture of its salts.1 A simple U-shaped cell, such as that used for moving-boundary electrophoresis (Chapter 9), with two ion-permeable membranes equidistant from the center, created a central compartment that separated anodic and cathodic chambers, as shown in Figure 11.1. Redox reactions occurring in the anodic (Eq. 11.1) and cathodic (Eq. 11.2) electrolyte compartments generated H+ and OH ions in the respective chambers ... 

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