Blotting of proteins

Western blotting has become an important, modern technique for analysis and characterization of proteins. The procedure consists of, first, the electrophoretic transfer (blotting) of proteins from polyacrylamide gels to synthetic membranes. The transferred blots are then probed using immunological detection methods to identify proteins of specific structure and/or function. In this experiment, bovine serum will be fractionated by SDS-PAGE and the proteins blotted onto a nitrocellulose membrane. Serum glycoproteins will be identified by their specific interaction with the lectin concanavalin A. 

Ranganathan, V. and De, P. K. (1995) Western blot of proteins from Coomassie-stained polyacrylamide gels. Anal. Biochem. 234, 102-104. 

Blotting of proteins can be viewed as the result of two operations (Gershoni and Palade, 1983) (i) their elution from the gel and, (ii) the immobilization of the eluted proteins on the membrane with the smallest possible loss in resolution. 

Western blotting of protein extracts of various C. roseus plant materials after SDS-PAGE, with polyclonal antibodies raised against the purified enzyme from elicited C roseus cells, revealed several bands. During the development of seedlings, first an immunoresponsive 54.8-kDa band, which is devoid of activity, is noted. Subsequently, a transient increase of a 55-kDa band is observed, which coincides with the occurrence of TDC activity. With the decrease of the TDC activity some other bands could be detected (40, 44, and 67 kDa). In leaves, the 54.8-kDa band could not be detected (178). In a series of in vitro experiments, Fernandez et al. (186) studied the stability of TDC. The presence of Mn, or particularly has a stabilizing... 

Both universal staining procedures and specific detection techniques can be performed after (electro) transfer or (electro)blotting of proteins (also called Western blotting) from the gel matrix (which sometimes hinders protein analysis) to a nitrocellulose or polyvinylidenedifluoride membrane, to which they are bound and immobilized. On the membrane, protein molecules are faster and better accessible for the interactions with the applied specific antibodies. The antigen-antibody complexes are visualized by a second antibody (against the first antibody) with an attached enzyme label, which catalyzes the color reaction in the place of the protein zone. 

WN. Burnette (1981) "Western Blotting" Electrophoretic transfer of proteins from Sodium Dodecyl Polyacrylamide gels to unmodified nitrocellulose and radiographic... 

The PemB cellular localisation was determined both in E. chrysanthenu and in an E. coli recombinant strain by Western blot of the cell fractions with a PemB-antiserum. No PemB was detected in the culture supernatant and only trace amounts were found in the soluble cell fractions - periplasm and cytoplasm (Figure 2). PemB was found mostly in the total membrane fraction from which it could be completely extracted by Triton X-100/Mg2+ and partially extracted by Sarkosyl (Figure 2). This behaviour is typical of inner membrane proteins, but since some exceptions have been noticed it does not positively indicate the PemB localisation (15). We performed cell membrane fractionation in sucrose density gradient centrifugation both by sedimentation and flotation, using several markers of inner and outer membrane vesicles. PemB was found in the outer membrane vesicles (data not shown). 

Specificity of the antisera was assessed by Western blotting. Electrophoretically separated proteins from culture filtrates were transferred to 0.45 fim nitrocellulose membranes. After transfer of proteins, membranes were... 

Because the protein analyte is endogenous to the plant, it can be difficult to demonstrate the efficiency of the extraction procedure. Ideally, an alternative detection method (e.g., Western blotting) is used for comparison with the immunoassay results. Another approach to addressing extraction efficiency is to demonstrate the recovery of each type of protein analyte from each type of food fraction by exhaustive extraction, i.e., repeatedly extracting the sample until no more of the protein is detected. " ... 

One of the first practical applications for these fluorescent labelled heparins was to examine the heparin binding behavior of different proteins and peptides under study in our laboratories. To this end we used a modification of the dot-blot assay described by Hirose and colleagues (13). F-D labelled heparin (-1 fluorescein/heparin) was radiolabelled with 125Iodine using iodobeads, to a specific activity of approximately 0.5 x 106 cpm/pg. Solutions of proteins with known heparin-binding capacities were dotted on nitrocellulose paper. A series of replicates... 

Figure 1.3 Western blotting of pRB protein extracted from two fresh cell lines, T24 and J82. The pRB proteins in fresh T24 cell line showed a stronger band than that obtained from J82 cell line. The Western blotting results correlated well with IHC staining intensity (Table 1.3 and Fig. 1.1). Reproduced with permission from Shi et al., Biotech. Histochem. 2007 82 301-309.

The use of independent methods, other than IFIC, for quantitative demonstration of proteins is particularly important. Both enzyme-linked immunosorbent assay (ELISA) and Western blot may be employed to confirm the amount of protein in a cell/tissue model, and in the protein-embedding bar code model under both comparable fresh and FFPE samples for accurate... 

Nirmalan NJ, Harnden P, Selby PJ, et al. Development and validation of a novel protein extraction methodology for quantitation of protein expression in formalin-fixed paraffin-embedded tissues using western blotting. J. Pathol. 2009 217 497-506. 

Various methods have been used to examine the composition of proteins adsorbed to SAMs. Overall adsorption patterns can be examined with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [50, 76, 77]. Absorbed proteins are eluted from the surface with surfactant (SDS), and then separated by electrophoresis. The proteins of interest are examined by western blotting [50, 76, 77]. Protein-specific antibodies can be used to detect proteins of... 

Unfortunately, there are no universal methods to detect all types of protein oxidation, because the products formed can be so diverse in nature. However, some forms of protein oxidation can be assayed using chemical modification (Davies et al., 1999 Shacter, 2000). In particular, the formation of carbonyl groups on proteins can be targeted using the reagent 2,4-dinitrophenyl-hydrazine (DNPH). This compound reacts with aldehydes to form 2,4-dinitrophenylhydrazone derivatives, which create chromogenic modifications that can be detected at high sensitivity in microplate assays or Western blot analysis (Buss et al., 1997 Winterbourn et al., 1999). 

Biotinylation of proteins on blots using the diazonium derivative of p-aminobenzoyl biocytin. 

Hydrazide groups can react with carbonyl groups to form stable hydrazone linkages. Derivatives of proteins formed from the reaction of their carboxylate side chains with adipic acid dihydrazide (Chapter 4, Section 8.1) and the water-soluble carbodiimide EDC (Chapter 3, Section 1.1) create activated proteins that can covalently bind to formyl residues. Hydrazide-modified enzymes prepared in this manner can bind specifically to aldehyde groups formed by mild periodate oxidation of carbohydrates (Chapter 1, Section 4.4). These reagents can be used in assay systems to detect or measure glycoproteins in cells, tissue sections, or blots (Gershoni et al., 1985). 

BLOTTING is a method to detect specific DNA (or RNA) fragments that contain sequences that are complementary to sequences in the labeled probe molecule. Only a few of the many DNA fragments on a gel will contain the sequence of interest, and only these will be seen (light up) on the blot. Specific proteins can also be visualized by blotting techniques using a specific antibody to detect a specific protein. 

If the gel separates DNA and the DNA is detected with a DNA probe, it is called a Southern blot. If RNA is separated on the gel and then detected by a DNA probe, it is a Northern. A Western uses specific antibodies to detect specific protein molecules on a blot of a protein gel. In the Western blot, the role of the DNA probe is filled by an antibody that recognizes a specific protein. 

Antonijczuk, K., O.S. Kroftova, A.H. Varghese, A. Antonijczuk, D.C. Henjum, G. Korza, J. Ozols, and W.F. Sunderman, Jr. 1995. The 40kDa 63Ni2+-binding protein (pNiXc) on Western blots of Xenopus laevis oocytes and embryos is the monomer of fructose-1,6-bisphosphate aldolase A. Biochim. Biophys. Acta 1247 81-89. 

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