Electroblotting from Polyacrylamide Gels

In some cases, stained blots are used only to identify protein band patterns while leaving the gel unmodified for subsequent steps (UNITB3.3). If such minimal protein transfer is desired, contact blotting is a suitable alternative. This unit also describes procedures for eluting proteins from membranes using detergents (Basic Protocol 2) or acidic extraction with organic solvents (Alternate Protocol 4). 

Polyacrylamide gel containing proteins of interest (unit 83.n 100% methanol 

Electroblotting apparatus solid plate electrode tank transfer system (e.g., Trans-Blot Cell, Bio-Rad) 

Gel support sheet (e.g., porous polyethylene sheet, Curtin Matheson) 

PVDF transfer membrane 0.2 p.m PVDF membrane (Bio-Rad) or Immobilon-P (Millipore) 

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Mozdzanowski, J., Hembach, P., and Speicher, D. 1992. High yield electroblotting onto poly-vinylidene difluoride membranes from polyacrylamide gels. Electrophoresis 13 59-64. 

Dunn, M.J. (1996b) Electroblotting of proteins from polyacrylamide gels, Methods Mol. Biol. 59, 363-3/0. 

The actual blotting process may be accomplished by one of two methods passive (or capillary) transfer and electroblotting. In passive transfer, the membrane is placed in direct contact with the polyacrylamide gel and organized in a sandwich-like arrangement consisting of (from bottom to top) filter paper soaked with transfer buffer, gel, membrane, and more filter paper. The sandwich is compressed by a heavy weight. Buffer passes by capillary ac-... 

Figure B3.2.2 Electroblotting with a tank transfer unit. The polyacrylamide gel containing the protein(s) to be transferred is placed on the smooth side of the polyethylene sheet (or filter paper sheets) and covered with the PVDF membrane and then a single sheet of filter paper. This stack is sandwiched between two fiber pads and secured in the plastic gel holder cassette. The assembled cassette is then placed in a tank containing transfer buffer. For transfer of negatively charged protein, the membrane is positioned on the anode side of the gel. Charged proteins are transferred electrophoretically from the gel onto the membrane.

Staining of blot transfer membranes permits visualization of proteins and allows the extent of transfer to be monitored. In the protocols described in this unit, proteins are stained after electroblotting from one-dimensional or two-dimensional polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes (unitb3.2). PVDF is the preferred, more universal membrane and is emphasized here however, most stains work similarly on nitrocellulose, and many can be used on alternative blotting membranes. 

Ogawa, H., et al. (1990). Direct Carbohydrate Analysis of Glycoproteins Electroblotted onto Polyvinylidene Difluoride Membrane from Sodium Dodecyl Sulfate Polyacrylamide Gel, Anal. Biochem. 190 165-169. 

Kyhse-Andersen, J. (1984). Electroblotting of multiple gels A simple apparatus without tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J. Biochem. Biophys. Methods 10, 203-210. 

FIGURE 3 N-terminal sequence analysis of protein samples recovered by electroblotting from SDS-polyacrylamide gel. The proteins are separated by SDS-PAGE and isolated by electroblotting onto PVDF membranes. The proteins are visualized by staining and excised from the blot for subsequent sequence analysis on a standard or specialized reaction cartridge. 

C. N-TERMINAL SEQUENCE ANALYSIS OF SAMPLES RECOVERED FROM SDS-POLYACRYLAMIDE GELS BY ELECTROBLOTTING... 

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 bound proteins are eluted from the beads by boiling in an SDS-PAGE sample buffer, electrophoresed on a 15% SDS-polyacrylamide gel, and electroblotted on to a PVDF membrane. 

Antigens are separated by polyacrylamide gel electrophoresis, electroblotted onto a membrane and then incubated with serum samples from subjects. Antigen-antibody reactions are detected by treatment of the strips with antiserum to human IgG (or other globulin) and a specific stain. Molecular weights of the antigenic components are obtained with a pre-stained protein standard (Laemmli 1970) (Fig. 3.5.6). 

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...

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