Electroblotting protocol

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... 

Conduct gel electrophoresis. Proceed with electroblotting (see Basic Protocol 1, steps 1 to 14). 

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. 

Fluorescamine, or 4-phenylspiro[furan-2(3//),l/-phthalan]-3,3,-dione, is used to introduce a fluorescent label on electroblotted proteins via reaction with free amines. Transferred proteins are visualized on blot transfer membranes with UV light. This stain can be very sensitive and can be used in conjunction with a second detection method such as immunoblotting (also see Basic Protocol 3). However, the protein is irreversibly modified because fluorescamine reacts with available amino groups (i.e., lysines and the protein N terminus if it was not previously blocked). 

Protocol 14 Isolation of Host-Cell Protein Impurities by Electroblotting... 

Several variations of the basic electroblotting procedure have been developed. Among these are a number of attempts to improve the original protocol, focusing on increasing the amount of protein transferred and retained on the membrane. 

Double blotting This procedure was developed to reduce false positives arising as a consequence of nonspecific binding of secondary antibodies. In this protocol the membranes obtained after dot blotting or electroblotting were blocked with nonfat milk as usual, incubated with a primary antibody and washed following conventional procedures. The... 

The protocols described are compatible with any current commercial automated sequencer that can accommodate both electroblotted samples and polypeptides applied from solution. The Blott Cartridge (Cat. No. 401096) is available from Applied Biosystems. Additional instruments used include a vacuum concentrator (Speed-Vac concentrator Model SSI or SS2, Savant Instruments, Inc.). 

In this protocol, amino acid analysis on the submicrogram level of proteins and peptides electroblotted onto PVDF as well as in solution is described. 

FIGURE 1 Schematic drawing of steps 2,3, and 5 of the hydrolysis protocol for electroblot-ted proteins. 

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