Ppsq 31a protein sequencer

To obtain the N-terminal sequence of cleaved rVRF1, the purified protein was digested using trypsin and electrophoresed on SDS-PAGE. The gel was transferred to PVDF membrane and the cleaved product (45 kDa) region of the membrane was excised. Approximately 100 pmol of the collected peptide (45 kDa) was determined using a PPSQ-31A protein sequencer (Shimadzu). Edman degradation was carried out for 6 cycles.

Molecular mass and amino acid sequence determination.
Two pmol of the active peptide were analyzed by matrix-assisted laser desorption ionization technique (MALDI-TOF; CDMX, Mexico) with an α-cyano-4-hydroxycinnamic acid matrix at Chemistry Institute, UNAM, facilities.
For amino acid sequence, one nmol of αD-FrXXA was analyzed by automated Edman degradation using a PPSQ-31A Protein Sequencer (Shimadzu Scientific Instruments; Tokio, Japan) by Dr. Fernando Zamudio at Biotechnology Institute, UNAM. Transcriptomic studies will be published.

Edman degradation was performed to determine the sequence of the peptide PM-7 using a PPSQ-31A protein sequencer (Shimadzu, Kyoto, Japan) according to the manufacturer’s standard protocols for GFD.

After SDS-PAGE, the protein band of AlgA was electro-transferred onto a polyvinylidene difluoride membrane (Imobulon; Millipore, Darmstadt, Germany). Amino acid sequences were determined with a PPSQ-31A protein sequencer (Shimadzu Corporation; Kyoto, Japan).

The observed molecular weights and purities of the samples were determined using an autoflex speed TOF/TOF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) in linear mode with α-cyano-4-hydrorycinnamic acid as the matrix. All procedures were conducted according to the manufacturer’s standard protocols, and the data were analyzed using the software package provided by the manufacturer.
The complete peptide sequences were determined by Edman degradation on a PPSQ-31A protein sequencer (Shimadzu, Japan) according to the manufacturer’s protocols.

N-terminal sequencing was performed on proteins translated from leaderless mRNAs to identify their translation initiation amino acid residues. To get the test proteins, leaderless genes fused with 6 × His tag were over-expressed under the control of their own promoters based on the expression vector pMV261, and then, respectively, transformed into mc²155 strain to obtain a series of derivative strains. The native promoter could hardly be induced, so two highly expressed leaderless genes MSMEG_4921 and MSMEG_6422 were selected. For protein purification, the two mc²155 derivatives were harvested at mid-exponential phase, followed by cell lysis using French press. The clear lysate was loaded onto a Ni²⁺-NTA affinity column for protein purification. Purified proteins were applied on a 15% SDS-PAGE and then transferred to PVDF blotting membrane for Edman sequencing using PPSQ-31A protein sequencer (Shimadzu, Japan). The strains and primers used are listed in Table 1 and Table S1, respectively.

We determined the intact masses of crotamine-like myotoxins of venom from one individual (CM04) using ESI-MS on an LCQ Fleet Ion Trap Mass Spectrometer. Amino-terminal sequencing of one crotamine-like myotoxin (RP-HPLC fraction 8) was determined by automated Edman degradation on a PPSQ-31A Protein Sequencer (Shimadzu, Tokyo, Japan).