Electroblotting to PVDF

Protein sample electroblotted to PVDF, nitrocellulose, or nylon membrane... 

Various alternative strategies have been reported for obtaining internal sequences in high yield from proteins separated by SDS gel electrophoresis. A number of approaches using electroblotting to PVDF membranes instead of nitrocellulose include blotting to PVDF/CNBr digestion/extraction/protease... 

Figure 3. Sequence data for a 20 residue experimental tryptic peptide. A 27 kDa protein was purified firom a whole cell extract of a human melanoma cell using high resolution 2D gels followed by electroblotting to PVDF. Spots derived from four gels were combined for in situ digestion. The estimated amount of protein present on the combined blots was about 20 pmol. The initial yield was 3.4 pmoles and the repetitive yield was 86%.

All sample proteins were run on either 12% or 14% pre-cast Novex 1.0 mm, 10 well gels under non-reducing conditions according to Laemmli (7). Samples were immediately electroblotted to Immobilon-P PVDF membrane using a semi-dry (MilliBlot-SDE) electroblotter essentially quantitiatively (8). After blotting, PVDF was washed briefly in HPLC water and stained with 0.05% Brilliant Blue-G Coomassie (BB-G) /20% methanol /0.5% acetic acid or Amido Black (2). The membrane was kept wet and not allowed to dry (9). Enzymatic digestion was performed as described (2) with all digestion and extraction buffer volumes reduced to 25 pL. 

Figure 2. Representative internal peptide sequence. In this experiment, a single band of HSA was electroblotted to Trans-6Iot PVDF (40 pmol loaded to gel), digested with trypsin, and separated by HPLC. The initial coupling was 2.8 pmoles and the repetitive yield was 88...

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... 

A representative analysis of bovine serum albumin electroblotted onto PVDF is given in Table I. The corresponding chromatogram is shown in Fig. 2. Under the standard conditions given, asparagine and glutamine are completely converted to aspartic acid and glutamic acid, respectively. Destruction of serine (up to 50%) and threonine (up to 30%) is usually expected. Cysteine and tryptophan are destroyed... 

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. 

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.

The electroblotting procedure for nitrocellulose membranes differs little from that for PVDF membranes. Nitrocellulose is compatible for use with a moderate amount of SDS (up to 0.1 %) in the transfer buffer. The delicate membranes must, however, be handled carefully and protected from high concentrations of organic solvents. 

An alternative to the tank transfer system is the semidry transfer system. In this procedure, the gel is stacked horizontally on top of the membrane in the transfer apparatus. Because only a small volume of transfer buffer is used, SDS from the gel is less effectively diluted, which may result in incomplete binding and lower yields, especially with PVDF membranes. For this reason, semidry transfer units are not recommended when reproducible high recoveries of electroblotted proteins are desired (e.g., for subsequent sequence analysis). Some procedures recommend stacking multiple transfer sandwiches to achieve several transfers simultaneously. To prevent unbound protein from migrating through the next gel and onto the membrane in the next transfer stack, sheets of porous cellophane sheets or dialysis membrane are placed between adjacent transfer stacks (see Fig. B3.2.3). Semidry electrotransfer requires shorter transfer times than tank transfer. 

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. 

Tovey, E.R. and Baldo, B.A. (1989) Protein binding to nitrocellulose, nylon and PVDF membranes in immunoassays and electroblotting, J. Biochem. Biophys. Methods 19, 169-183. 

Figure 1. HPLC peptide map comparisons (215 nm) using different membrane types. In situ tryptic digestion of replicate apomyoglobin bands (ICX) pmols loaded/lane) electroblotted from a single gel onto 2—Nitrocellulose, 2—Immobilon P, J—Trans-Blot PVDF. B—peaks in a trypsin/buffer control chromatogram. P—PVP-40 peak which is variable from run to run, but more prominent on Trans-Blot membranes.

Electroblotting was conducted using a semIdry-type electroblotter AE-6670 (Atto). Proteins transferred to the PVDF membrane were stained with 0.1% (w/v) Coomassie brilliant blue R-250 (CBB) in methanol/acetic acid/water (5 2 5, v/v) for 5 min and destained with 90% (v/v) methanol. The stained band was cut out, and... 

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