反应详情

For N-terminal sequence analysis and Western blotting, proteins were transferred onto a PVDF membrane (Problott, Applied Biosystems) using a transblot cell (Bio-Rad). The PVDF membrane was wetted in 100% methanol and soaked in transfer buffer (10 mM CAPS, 10% v/v methanol, pH 11.5). Transfer was performed using a potential difference of 60 V for 90 min. For N-terminal sequencing membranes were stained with 0.1% (w/v) Coomassie brilliant blue R250 in methanol/water/acetic acid for 30 sec and destained in 50% v/v methanol. Protein bands were excised and N-terminal sequences determined using a Hewlett Packard 10005A protein sequencer. For peptide mass fingerprinting analysis; Coomassie blue stained protein bands from SDS-PAGE were excised and subjected to in-gel trypsin digestion and subsequent peptide extraction. Protein bands were excised from the Coomassie Blue stained SDS-PAGE gel and gel pieces were washed in 50 mM NH4HCO3/ethanol 1:1, reduced and alkylated with DTT and iodoacetamide, respectively and digested with sequencing grade modified trypsin (Promega) overnight at 37° C. as previously published Mortz et al. (1996). Electrophoresis 17:925-31]. The peptide extract containing 25 mM NH4HCO3 was then analysed by MALDI-TOF MS using an Ultraflex TOF/TOF instrument (Bruker Daltonics) in positive ion and reflectron mode. A saturated solution of 4-hydroxy-α-cyanocinnamic acid (HCCA) was prepared in 97:3 v/v acetone/0.1% v/v aqueous TFA. A thin layer was prepared by pipetting and immediately removing 2 μL, of this solution onto the 600 μm anchorchips of the target plate. Sample (0.5 μL) was deposited on the thin layers with 2.5 μL of 0.1% v/v aqueous TFA, and allowed to adsorb for 5 min, after which the sample solution was removed, and the thin layers washed once with 10 μL of ice-cold 0.1% v/v aqueous TFA for 1 min. Spectra were calibrated by close external calibration using a standard peptide mix. Proteins were identified by peptide mass fingerprinting against the P. gingivalis database (available from www.tigr.org) using an in-house Mascot search engine. Table 2 shows the peptide sequences used to identify the SDS-PAGE separated protein bands of the antigenic complex. The SDS-PAGE of the complex (FIG. 2) is annotated with the designation of the proteins identified by N-terminal sequencing and peptide mass fingerprinting. The complex was found to consist of: Kgpcat, RgpAcat, RgpAA1, KgpA1, RgpAA3, RgpAA2, KgpA2, HagAA1*, HagAA1**, HagAA3 and HagAA2 as well as partially processed Kgp (residues 1 to 700 and residues 136 to 700). A schematic of the processed domains of RgpA, Kgp and HagA are shown in FIG. 3.

1. customfor 90 min
2. extractionsubjected to in-gel trypsin digestion and subsequent peptide extraction
3. washwere washed in 50 mM NH4HCO3/ethanol 1:1
4. custommodified trypsin (Promega) overnight at 37° C. as previously published Mortz et al. (1996)
5. extractionThe peptide extract
6. additioncontaining 25 mM NH4HCO3
7. customA thin layer was prepared by pipetting
8. customimmediately removing 2 μL
9. customafter which the sample solution was removed
10. washthe thin layers washed once with 10 μL of ice-cold 0.1% v/v aqueous TFA for 1 min
11. customby close external calibration
12. additionmix
13. customseparated protein bands of the antigenic complex