Proteins separated by electrophoresis and electroblotted onto Immobilon-P Transfer Membrane can be sequenced directly in the gas-phase sequencer. Protein bands visualized by Coomassie Blue are placed in the sequencer cartridge without the addition of polybrene. Preconditioning sequencer cycles are eliminated, reducing reagent use and instrument operating time. The average initial yield for protein spotted or blotted onto the polyvinylidene-based membrane was determined to be 70 to 80% using 125I-labeled beta-lactoglobulin. Preliminary data indicate that proteins hydrolyzed in situ on Immobilon-P can further be characterized by amino acid compositional analysis.