Pharmacia Biotech Multiphor II User Manual page 44

Immobilizing membranes
The immobilizing membrane is an important factor affecting the efficiency
of the electrophoretic transfer. The most important requirements for an
immobilizing membrane are:
• High binding capacity for the molecules of interest
• Preservation of the biologic activity of the molecules of interest
• No interference with subsequent detection methods
• Chemical and mechanical stability to assay conditions
• Provision of an accurate reflection of the original separation
Nitrocellulose membranes are the standard medium for electrophoretic
transfer of proteins and nucleic acids. This is due to their high binding
capacity, versatility and easy use. Nitrocellulose membranes are available in
various pore sizes, 0.45 µm is most commonly used, however low molecular
proteins may be lost. By using pore sizes of 0.2 or 0.1 µm, most proteins
are retained.
Nitrocellulose membranes can be probed several times. The membranes
require no activation and the functional groups have an extended lifetime.
Protein patterns on nitrocellulose membranes can be easily detected with
most conventional stains, as well as by autoradiographic, immunoenzymatic
and fluorescent methods.
Other membranes are: nylon-based membranes, diazobenzyloxymethyl
(DBM) and diazophenylthioether (DPT) papers.
Transferring proteins from SDS polyacrylamide gels to
nitrocellulose membrane.
The support (or backing) film must be removed from all polyacrylamide and
agarose gels before electrophoretic transfer. Using FilmRemover, the film is
removed quickly and cleanly. Instructions for use are supplied with
FilmRemover.
Artisan Technology Group - Quality Instrumentation ... Guaranteed | (888) 88-SOURCE | www.artisantg.com
5. Operation
41