Self-Dimerization and Alanylation Assay

1000 pmol of P1c2 or P1c2^UGGU was dissolved in 2 µL of ACCA reaction buffer. After heating at 95 °C for 10 min, it was incubated on ice for 60 min to be self-dimerized. Then, 1000 pmol of alanyl-minihelix^Ala was added and placed at 4 °C for 24 h. The reaction product was treated with 0.22 µL of 250 mM KOH (pH of the solution was changed from 7.5 to pH 9.0) at room temperature for 45 min to liberate any alanylalanine that might form. As a negative control, 2 µL of ACCA reaction buffer was used instead of 2 µL of 1000 pmol of P1c2 or P1c2^UGGU in the first step.

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Kawabata M., Kawashima K., Mutsuro-Aoki H., Ando T., Umehara T, & Tamura K. (2022). Peptide Bond Formation between Aminoacyl-Minihelices by a Scaffold Derived from the Peptidyl Transferase Center. Life, 12(4), 573.

Publication 2022

Alanylalanine Buffer

Corresponding Organization : Tokyo University of Science

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Variable analysis

independent variables

Concentration of P1c2 or P1c2^UGGU (1000 pmol)
Concentration of alanyl-minihelix^Ala (1000 pmol)
Treatment with 0.22 µL of 250 mM KOH (pH change from 7.5 to 9.0)

dependent variables

Self-dimerization of P1c2 or P1c2^UGGU
Formation of alanylalanine

control variables

Volume of ACCA reaction buffer (2 µL)
Temperature (95 °C, 4 °C, room temperature)
Incubation times (10 min, 60 min, 24 h, 45 min)

positive control

2 µL of ACCA reaction buffer instead of 1000 pmol of P1c2 or P1c2^UGGU

negative control

None mentioned

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