Electroblotting protein preparation

Fig. 5 shows the representative sequence data of carbonic anhydrase fria ent 5 from the sample prepared by SDS-PAGE/electroblotting approach. A major sequence of PALKPLALVYGEATS...(starting from residue 41 of the protein) could be identified without difficulty. Another sequence of LKFR IT.NFNAEGEPE... was also found in this sample. 

Hsieh, J., et al. (1988). Electroblotting onto Glass-fiber Filter from an Analytical Isoelectrofocusing Gel A Preparative Method for Isolating Proteins for N-terminal Sequencing, Ancd. Biochem. 170 1-8. 

Multiple tissue Western (MTW) blot Human MTW blots provide a new immunological tool for the investigation of tissue-specific protein expression. These are premade immunoblots prepared using proteins isolated from adult human tissue. The proteins are isolated from whole tissue under conditions engineered to minimize proteolysis and to ensure maximal representation of tissue-specific proteins. SDS solubilized proteins are fractionated by SDS PAGE and electroblotted onto PVDF membranes to generate blots ready for incubation with... 

Fig. 2. Western blot analysis of protein bound to immunoabsorbant columns. PyBHK EF-2 preparations containing cellular ADP-ribosyltransferase activity were chromatographed on Sepharose 4B coupled to Pseudomonas toxin A antibody or pyBHK ADP-ribosyltransferase antibody. After washing unbound material from the resin with PBS, the bound protein was eluted from the columns with 1 M propionic acid, concentrated under vacuum and subjected to electrophoresis on a 7.5% SDS-polyacrylamide slab gel. In addition. Pseudomonas toxin A standards were subjected to electrophoresis on the gel. Following electroblotting of the proteins from the ge) to a nitrocellulose sheet, the sheet was sectioned and reacted with Pseudomonas toxin A antibody (A-Q or with pyBHK ADP-ribosyltransferase antibody iP-F). Pseudomonas toxin A (A) protein from Pseudomonas toxin A antibody coupled immunoabsorbant B) protein from pyBHK ADP-ribosyltransferase antibody coupled immunoabsorbant (C, D) protein from Pseudomonas toxin A antibody-coupled immunoabsorbant (i ) Pseudomonas toxin A F). The numbers represent Mj. X 10" of the mol.wt. standards...

Aebersold R.H., Teplow D.B., Hood L.E., and Kent S.B. 1986. Electroblotting onto activated glass. High efficiency preparation of proteins from analytical sodinm dodecyl sulfate-polyacrylamide gels for direct sequence analysis, /. Biol. Chem., 261 4229-4238. 

The initial steps of solubilization, DEAE chromatography and gel filtration were slight modifications of reported procedures [19]. The pooled fractions from a Sephacryl S-200 (Pharmacia) column were applied to a Mono Q HR5/5 FPLC column (Pharmacia) and eluted in a 20 ml gradient from 0-350 mM NaCl in 0.25 M sucrose-20 mM Tris-HCl pH 7.3. Fractions of 1 ml were collected and assayed for binding of NAA-1-[ C] (61 mCi/mmol, Amersham) by one of three methods [20]. The most active fractions were pooled, desalted and lyophilized. This preparation (approx. 50% receptor) was used either for monoclonal antibody production or was fully purified by native PAGE in a neutral pH discontinuous system [3]. The gel was briefly electroblotted (5 min, 10 mA) to nitrocellulose and the small fraction of transferred proteins visualized by rapid staining [8]. This blot was then used to locate precisely the bulk protein bands remaining in the gel. 

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