Protein blots

B. A. Baldo and E. R. Tovey, Protein Blotting. Methodology, Kesearch, and Diagnostic Applications, Karger, Swit2edand, 1989. 

In order to reduce the time required to confirm the accumulation of a given recombinant protein, we have developed a cell culture system in which transgenic alfalfa callus material produced at the proliferation step of Agrobacterium-based transformation is used to initiate cell cultures. These cell suspensions can be subcultured to sustain batch production of modest protein amounts. The protein blot shown in Fig. 1.2 demonstrates our ability to detect a recombinant protein in total... 

Fig. 1.2 Protein blot analysis of human therapeutic protease inhibitor (HTPI) produced in alfalfa cell cultures using different promoters and subcellular targeting peptides as shown. Equal amounts of total soluble proteins from cell cultures were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a polyvinyldifluoride (PVDF) membrane. Monoclonal anti-HTPI IgGs were used for detection.

Fig. 1.4 Protein blot analysis of C5-1 assembly in agroinfiltrated alfalfa leaves. Total leaf soluble proteins, extracted 4 days after infiltration were separated by SDS-PAGE under non-reducing conditions and blotted onto a PVDF membrane. Polyclonal antimouse IgGs were used for detection. Purified C5-1 was mixed with total soluble proteins from control infiltrated alfalfa leaves and loaded as a standard.

Baldo, B.A. and Tovey, E.R. (Eds.), Protein Blotting Methodology, Research and Diagnostic Applications, Karger, Basel, 1989. 

Dunbar, B.S. (Ed.), Protein Blotting A Practical Approach, Oxford University Press, Oxford, 1994. 

Human CYP46 in brain microsomal fraction by RNA and protein blots (Lund et al,... 

Proteins blotted on PVDF membranes are stainable with Coomassie Brilliant Blue R250, but a relative intense background remains, which does not influence, for example, amino acid sequence analysis. 

Proteins blotted on PVDF can be subjected directly to sequence analysis. Specialized protocols for this purpose are referred to, for example, by JuNGBLUT (1997). 

On-blot digestion is another method used for proteins blotted onto nitrocellulose or PVDF membranes. Procedure is similar to in-gel digestion with the exception of elution from the membrane surface. 

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. 

B. Dunbar, Protein Blotting, A Practical Approach (1994), IRL Press (Oxford). Complete and up-to-date book on blotting. 

Use of Proteins Blotted to Polyvinylidene Difluoride Membranes as Immunogens... 

Szewczyk, B. and Summers, D. F (1988) Preparative elution of proteins blotted to Immobilon membranes. Anal. Biochem 168,48-53. 

Nylon membranes are not recommended for protein blotting, since they almost always suffer from high background staining. 

Proteins, blotted from polyacrylamide gels onto nitrocellulose sheets (Western blots) can be stained nonspecifically with a variety of dyes, or they can be identified individually by probing with appropriate antibodies. These procedures may be performed on duplicate blots, staining the total protein pattern on one blot and using the second blot for the immune reaction (1,2) This chapter describes how to combine both methods on one blot, i.e., staining the blot first for total protein, followed by an indirect immune reaction (3). 

Rohnnger, R. and Holden, D W (1985) Protein blotting detection of protein with colloidal gold, and of glycoproteins and lectins with biotin-conjugated and enzyme probes Anal Biochem. 144, 118-127. 

Gershom, J M. and Palade, G E (1983) Protein blotting-principles and applications. Anal Biochem 131, 1-15... 

Vandekerckhove, J., Bauw, G.. Puypi, M., Van Damme, J., and Van Montagu, M. 1985. Protein-blotting on Polybrene-coated glass-fiber sheets. Eur. J. Biochem. 152 9-19. 

Moeremans, M., Daneels, G., and De Mey, J. 1985. Sensitive colloidal metal (gold or silver) staining of protein blots on nitrocellulose membranes. Anal. Biochem. 145 315-321. 

Alkaline hydrolysis, see Saponification Alkaline phosphatase (AP), in immunoblotting, 207-217 Amido black, staining of proteins blots, 199-200 Amino acids... 

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