2D SDS-PAGE

D-SDS-PAGE (2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis)... 

D-SDS-PAGE allows separation into smaller fractions whereas 2D-SDS-PAGE leads to separation into many fractions. Resultant fractions are used for digestion or further separation prior to MS analysis. 

The techniques of 1D and 2D SDS-PAGE (see Methods in Molecular Biology, Volumes 1 and 3), electroblotting (see Methods in Molecular Biology, Volume 3), and lmmunoblottmg (see Chapter 20) are described elsewhere. 

Figure 15.6 Schematic depiction of one example of protein identification by mass spectrometry. Genes of interest are tagged, then transfected into mammalian cells, and proteins associated with the cognate tagged protein are purified by affinity methods. Separation of the complex is carried out by 2D SDS-PAGE. Identification of the proteins is by MALDI and ESI (Blackwell, 1999).

D SDS-PAGE which combines tw o modes of separation isoelectric focusing and polyacrylamide gel elctrophoresis has been on the market for about 30 years (OTarrell, 1975 Barritaultetal., 1976). Initially this technique didnot gain much of popularity due to obstacles in part originating from poor... 

VGF polypeptide, unknown 7.7kDa polypeptide Apolipopiotein Al, apolipopiotem E, apolipopio- 2D SDS-PAGE and MALDl-TOF (Puchades et al., 2003)... 

Creutzfeldt-Jacob disease 14-3-3 pi otem cystadn C 2D SDS-PAGE,. SELDl-TOF and (Hairmgton et al., 1986 Sanchez... 

Multiple sclerosis Cartilage acidic pi otem, teti anecdn,. SPARC-hke 2D SDS-PAGE and LC -MS/MS (Hammack et al., 2004)... 

Fig. (2). Strategy for the identification of gel-separated (2D-SDS-PAGE) proteins that are part of the immune response of Drosophila. Blood from control and immune-challenged flies was collected and subjected to 2D-SDS-PAGE, immune-induced (-I-) or repressed (-) stained spots were excised, subjected to proteolysis e.g. trypsin), and the peptides fragments analyzed by MS and/or MS/MS.

D SDS-PAGE Morph/warp-stained Relative intensities of... 

The 2D-SDS-PAGE separation described results in a planar array of separated compounds. If these are unknown proteins (perhaps just one or a few which interrupt a standard pattern, and may signal a mutant form or an abnormal biological state), the spots of interest may be cut out or dissolved out of the gel. The isolated spot can be subjected to tryptic digest, the peptide digest mixture separated by capillary HPLC, and the separated peaks characterized, and if necessary, sequenced by ESI-MS and ESI-MS-MS, as was illustrated in Figs. 13.21 and 13.22. If the 2D separation is of peptides from a single protein in a... 

The protein kinase was isolated from the supernatant by affinity chromatography on ATP-Sepharose followed by gel filtration.Purity was tested by 2D SDS-PAGE and silver staining and the final isolate proved to contain practically only a single protein. 

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