Electroblotting

PVDF-based microporous filters are in use at wineries, dairies, and electrocoating plants, as well as in water purification, biochemistry, and medical devices. Recently developed nanoselective filtration using PVDF membranes is 10 times more effective than conventional ultrafiltration (UF) for removing vimses from protein products of human or animal cell fermentations (218). PVDF protein-sequencing membranes are suitable for electroblotting procedures in protein research, or for analyzing the phosphoamino content in proteins under acidic and basic conditions or in solvents (219). 

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. 

O Shannessy, D.J., Voorstad, P.J., and Quarles, R.H. (1987) Quantitation of glycoproteins on electroblots using the biotin—Streptavidin complex. Anal. Biochem. 163, 204-209. 

D. Schleuder, F. Hillenkamp, and K. Strupat. IR-MALDI-Mass Analysis of Electroblotted Proteins Directly from the Membrane Comparison of Different Membranes, Application to On-membrane Digestion, and Protein Identification by Database Searching. Anal. Chem., 71(1999) 3238-3247. 

Whereas gels are mainly electroblotted immediately after electrophoresis, it is possible to blot Coomassie or Amido Black stained gel too, but with lower efficiency and after soaking in ddH20 for 15 min and an equilibration step in transfer buffer C (see below) for 30 min. 

Kemper, C. Berggren, K. Diwu, Z. Patton, W. F. An improved, luminescent europium-based stain for detection of electroblotted proteins on nitrocellulose or polyvi-nylidene difluoride membranes. Electrophoresis 2001, 22(5), 881-889. 

Amido black dye Two years later, Grassman and Hanning developed another organic dye to be used on filter paper after electrophoresis amido black stain. It has moderate sensitivity. Today, amido black dye is used for colorimetric determination of electroblotted proteins on PVDF (poly-vinylidene difluoride) and nitrocellulose membranes. 

Stains in the form of colloidal dispersions The need for highly sensitive detection methods for proteins after blotting (e.g., electroblotting, dot-blotting, slot blotting) applied to nitrocellulose or PYDF membranes drove scientists attention to develop new stains. It was discovered that the stains in the form of colloidal dispersions are suitable for such applications. The most commonly used colloidal stains are... 

Gel Electrophoresis. This is becoming a more commonly used procedure for purifying proteins, nucleic acids, nucleoproteins, polysaccharides and carbohydrates. The gels can be electroblotted onto membranes and the modem procedures of identifying, sequencing (proteins and nucleic acids) and amplifying (nucleic acids) on sub-micro scales have made this technique of separation a very important one. (See D.Patel Gel Electrophoresis, J.Wiley-Lis, Inc., 1994). 

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-... 

Period 1 Fractionation of protein mixture by SDS-PAGE begin passive blot procedure or electroblotting. 

Begin electroblotting. Apply 20 V for overnight runs. It is not necessary to use methanol in the transfer buffer, since it does not improve the binding of proteins to this membrane (see Note 3). 

Lauriere, M (1993) A semidry electroblotting system efficiently transfers both high-molecular-weight and low-molecular-weight proteins separated by SDS-PAGE.Anal Biochem 212,206-211... 

The techniques of 1D and 2D SDS-PAGE (see Methods in Molecular Biology, Volumes 1 and 3), electroblotting (see Methods in Molecular Biology, Volume 3), and lmmunoblottmg (see Chapter 20) are described elsewhere. 

Basic Protocol 1 Electroblotting onto PVDF Membranes B3.2.1... 

Alternate Protocol 1 Electroblotting of Proteins for Sequence Analysis B3.2.5... 

Alternate Protocol 2 Electroblotting onto Nitrocellulose Membranes B3.2.6... 

Alternate Protocol 3 Protein Electroblotting in Semidry Systems B3.2.7... 

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

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.

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