Trypsin profile igd kit

Reactive Lys residues available for chemical coupling of antigens were determined by FITC-labeling free amino groups, FITC-modified coat protein was separated in SDS/PAGE gel, the fragment was excised from gel and digested with trypsin (Trypsin Profile IGD Kit, Sigma). VLPs (1.5 mg/ml in 50 mM Sodium borate, 2 mM EDTA, pH 9.0) were reacted with 20 mM FITC at +4 ^oC for 24 h. Samples after FITC treatment were separated in 12% SDS-PAGE, protein spots after Coomassie staining excized from gel and treated with Trypsin using Trypsin Profile IGD Kit and protocol (Sigma, St Louis, USA) ON +4 ^oC. The reaction mixture was purified using ZipTip tips (Merck Millipore, Cork, Ireland), diluted with a 3-hydroxypicolinic acid matrix solution and spotted onto an MTP AnchorChip 400/384TF. MALDI-TOF MS analysis was carried out on an Autoflex MS (Bruker Daltonik, Bremen, Germany). The protein molecular mass (MM) calibration standard I (3–20 kDa; Bruker Daltonik) and Peptide calibration standard II was used for mass determination.

Protein bands were excised from the rDer p 18 preparations which had been separated by SDS-PAGE and were digested using the Trypsin Profile IGD Kit (Sigma-Aldrich, St. Louis, MO, USA). The Nano LC-ESI MS/MS data were acquired and analysed as described [27 (link)] except that generated peak lists were searched against the Swiss Prot databank using MASCOT (Matrix Science) search engine.

SM was made available by the German Ministry of Defense and integrity as well as purity was proved by NMR in house. Allylisothiocyanat (AITC), iodoacetamid (IAA), ethanol (EtOH), glycerol, glycine, trifluoroacetic acid (TFA), acetonitrile (ACN), penicillin/streptomycin (P/S) and Trypsin Profile IGD Kit for proteolysis of protein spots were obtained from Sigma-Aldrich (Steinheim, Germany). 2D-Clean-UP Kit, Silver Staining Kit (protein), 2D-Quant-Kit and Coomassie Brilliant Blue Solution were obtained from GE Healthcare (Freiburg, Germany). Bromophenol blue and sodium dodecyl sulfate (SDS) were purchased from Bio-Rad (Munich, Germany). RT² First Strand Kit, RT² SYBR Green/ROX qPCR Mastermix, RNeasy Protect Mini Kit and RT² Custom Profiler PCR 96-well plates with specific customized primers were purchased from QIAGEN Sciences (Venlo, The Netherlands). Dulbecco’s minimal Eagle medium (DMEM), fetal bovine serum (FBS), trypsin–EDTA (ethylenediaminetetraacetic acid) and phosphate-buffered saline (PBS) were obtained from Life Technologies (Gibco, Karlsruhe, Germany). AP18 was delivered by Bio-Techne (Wiesbaden-Nordenstadt, Germany). α-cyano-4-hydroxycinnamic acid (CHC) as matrix for MALDI-TOF measurements was obtained from Bruker Daltonics (Bremen, Germany).