Comparative gas phase and pulsed liquid phase sequencing on a modified Applied Biosystems 477A sequencer

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• The Applied Biosystems 477A sequencer is a widely used instrument for protein sequencing.
• Sequencing on polyvinylidene difluoride (PVDF) membranes presents unique challenges.
• Existing protocols may require time-consuming steps and additional reagents.

Purpose of the Study:

• To describe a simple and inexpensive modification of the Applied Biosystems 477A sequencer.
• To evaluate the performance of the modified sequencer in both pulsed liquid-phase and gas-phase modes.
• To highlight the advantages of the gas-phase mode for sequencing on PVDF membranes.

Main Methods:

• Modification of the Applied Biosystems 477A sequencer.
• Comparison of pulsed liquid-phase and gas-phase sequencing protocols.
• Analysis of sequencing performance using samples on glass fiber filters and PVDF membranes.

Main Results:

• The gas-phase mode eliminates the need for additional carriers when sequencing on PVDF membranes.
• Gas-phase sequencing reduces time-consuming preconditioning cycles and lowers sequencer background.
• Initial coupling yields were higher in the gas-phase mode compared to the pulsed liquid-phase mode.
• Average repetitive yields were comparable between the two modes.

Conclusions:

• The described modification provides a versatile and efficient method for protein sequencing.
• The gas-phase mode offers significant advantages for sequencing samples immobilized on PVDF membranes.
• This approach simplifies the sequencing workflow and improves data quality.