Each preparation was separated into a soluble stromal and membrane fraction and then resolved by 1D SDS-PAGE, followed by in-gel trypsin digestion and identification by online nano-liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) with an LTQ-Orbitrap, using data dependent acquisition (DDA) and dynamic exclusion.
An equal amount of protein (30 µg/sample) of each tissue extract preparation was separated on 14% SDS-PAGE, followed by immunoblot analysis with rabbit anti-human cathepsins S, L, and K polyclonal antibodies (1∶1000, Calbiochem, San Diego, CA) or rabbit anti-human cystatin C polyclonal antibodies (1∶1000; DAKO Corp., Carpinteria, CA), all of which cross-reacted with mouse gene products.
For immunoblot analysis, an equal amount of protein from each cell type preparation was separated by SDS PAGE, blotted, and detected with mAbs against mouse FcεR1α (1:1,000; eBioscience, Inc).
A 2 μL aliquot of the resuspended total RNA preparation was separated on a 1% agarose gel to confirm that the RNA was not degraded, and the RNA quantity and purity were measured with a NanoDrop ND-1000 Spectrometer (NanoDrop Technologies).
The concentrated preparation was separated by gel filtration on a Sephadex G-100 superfine column (2.6 × 85 cm) equilibrated with 10 mM phosphate (pH 8.1) containing 2 μM PLP and 1 mM 2-ME (buffer A).
Each glycan preparation was separated using a 1.7 μm BEH glycan column (2.1 mm×150 mm, Waters) and analysed by UHPLC FLD MS using a Waters ACQUITY UPLC® H-Class Bio with fluorescence detection coupled to a Waters Xevo G2-S Q-ToF mass spectrometer.
Mitochondrial preparations were separated on standard 12% glycine SDS polyacrylamid gels.
LPS preparations were separated by SDS-PAGE using a 4% polyacrylamide stacking gel and a 15% polyacrylamide separating gel.
Briefly, intact borrelial cells were incubated with either proteinase K or trypsin and whole-cell protein preparations were separated by SDS-PAGE (13%) as described [26].
