Protein 2D-PAGE

For proteins of low abundance as well as membrane proteins, 2D PAGE suffers from a lack of resolving power. Another possibility, besides chromatography, to circumvent the limitations of 2D PAGE is to use chemically labeled tags, such as two different isotopes (ICAT, isotope-coded affinity tag) (Adam, 2002). A suitable... 

Proteins 2D-PAGE/MS, protein chips, enzymatic analyses, immunologic methods... 

Nucleotides Isotopomer distributions in intracellular proteinogenic amino acids Proteins, 2D-PAGE... 

D-polyacrylamide gel electrophoresis) maps of protein mixtures is discussed. 2D PAGE is considered the classical and principal tool for protein separation—prior to mass spectrometry—to achieve the main goal of proteomics, that is, a comprehensive identification and quantification of every protein present in a complex biological sample that would allow analysis of an entire intact proteome (Wilkins et al., 1997 Righetti et al., 2001 Hamdan and Righetti, 2005). 

The major advantage of 2D-PAGE is that it enables simultaneous separation of thousands of unknown proteins, first by charge using isoelectric focusing (IEF) and... 

Moreover, experimental reference maps of human tissues were studied p7 and Mr coordinates of identified spots were retrieved from the SWISS-2D-PAGE database, the values <3X = 0.009 pH and av = 0.0002 log Mr were assumed for spot dimension since they represent the standard case for experimental 2D-PAGE maps—normal sample loading of a tissue homogenate (ca. 1 mg total protein) and standard gel sizes (18 x 20 cm, IEF x SDS-PAGE). 

It must be emphasized that the availability of the SMO and 2D autocovariance function methods as two independent statistical procedures to estimate the same parameter, in, the number of proteins, is a helpful tool to verify the reliability of the results obtained. In the case of the 2D PAGE map of colorectal adenocarcinoma cell line (DL-1) an excellent agreement was found between the values obtained from the SMO method—m = 101 10 and m = 105 10—and the 2D autocovariance function procedure—m = 104 10 (Pietrogrande et al., 2006a). 

Dondi, F., Bassi, A., Cavazzini, A., Pietrogrande, M.C. (1998). A quantitative theory of the statistical degree of peak overlapping in chromatography. Anal. Chem. 70, 766. Eckerskom, C., Strupat, K., Schleuder, D., Hochstrasser, D.F., Sanchez, J.-C., Lottspeich, F., Hillenkamp, F. (1997). Analysis of proteins by direct-scanning infrared-MALDI mass spectrometry after 2D-PAGE separation and electroblotting. Anal. Chem. 69, 2888. Expasy,http //www.expasy.ch. 

FIGURE 4.1 2D polyacrylamide gel electrophoresis (2D-PAGE) maps of protein mixtures. (See text for full caption.)... 

Cerfain proteins were confirmed by Western blot analysis. The 2D PAGE analysis can resolve up to 2000 proteins. In this study, 820 spots were mapped to proteins but only 165 were used in the protein-to-gene analysis. Presumably these represent the highest fold expressed genes from the Ixmg tumors that were also visible on the gels by silver staining. 

After finishing the 2D-PAGE, proteins are separated both by their isoelectric points (pi) and molar masses (Mr). 

Fig. 2A, B Global (A) vs targeted (B) proteomics. In a global proteomics approach, all the protein constituents of a cell are isolated and subjected to 2D PAGE. In a targeted proteomics experiment, only a subset of proteins is analyzed. An example is the complex that is formed between a transmembrane receptor and cytoplasmic signaling proteins upon ligand activation of the receptor...

Fig. 3A, B Strategies for the analysis of complex protein mixtures. A Proteins are separated by 2D PAGE and the relevant protein spots excised from the gel, digested, and analyzed by mass spectrometry. B Proteins are first digested into small peptides and then fractionated by two rounds of chromatography before each peptide is analyzed by mass spectrometry...

Although almost 30 years old, the most widely used technology in comparative proteomics is still 2D PAGE. This applies to the global analysis of proteins... 

However, analysis of protein mixtures derived from cells, tissues, and body fluids by 2D PAGE by no means represents a comprehensive picture of the proteins in the mixture. Proteins with extreme isoelectric points, large proteins, small proteins, and hydrophobic proteins are commonly not amenable to 2D PAGE and hence can be easily missed. Furthermore, low abundant proteins are often not detected in 2D gels when proteins of high abundance are present. This limitation is particularly relevant when analyzing serum or other body fluids, where protein amounts vary by ten orders of magnitude (Anderson and Anderson 1998). 

Fig. 5 2D PAGE of CSF before and after depletion with Cibacron blue/protein G affinity resins...

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