Spinx centrifuge filter

The monodispersity of the purified TAP complexes was analyzed by multicolor fluorescence-based size exclusion chromatography (MC-FSEC)27 (link). The fluorescent proteins fused to TAP subunits (mVenus on TAP1 and mCerulean on TAP2) or ICP47 were detected by an Agilent 1200 series high-performance liquid chromatography (HPLC) system using a Shodex semi-micro KW404-4 F (4.66 × 300 mm) column. The running buffer was composed of 20 mM HEPES/NaOH pH 7.4, 200 mM NaCl, 50 mM KCl, 5% glycerol, and 0.05% GDN. For the MC-FSEC analysis, 60 μl of the purified TAP complexes were filtered through 0.22 μm SpinX centrifuge filter (Costar). 50 μl of each sample were then injected by an autosampler. To determine the thermostability of the ICP47-TAP complexes, 60 μl of the protein solution were incubated for 1 h at 40 °C, then treated as described above, and compared by MC-FSEC with a sample stored for 1 h on ice. The melting points of the protein complexes were determined by incubating samples five min at 25–50 °C. Elution profiles were overlaid and the overlap area was calculated with ImageJ 1.48 v (Fig. 4c). The area of the profiles at 4 °C was normalized to 100%. Curve fit was calculated with GraphPad Prism 5 using the nonlinear regression “EC50 shift fit”.

Halo-NEMO resin [24 (link)] was washed twice with 6 M urea solution and five times with 50 mM Tris–HCl pH 7.5, 1% (v/v) Triton X-100. Following incubation overnight with cell extract (2 mg per 20 µl packed Halo-NEMO resin), the resin was collected by centrifugation, washed twice with 50 mM Tris–HCl pH 7.5, 500 mM NaCl, 1% (v/v) Triton X-100 and twice with 50 mM Tris–HCl pH 7.5, 1% (v/v) Triton X-100 and the supernatant discarded.
The packed beads were resuspended in 50 mM HEPES pH 7.5, 100 mM NaCl, 2 mM dithiothreitol, 0.01% (w/v) Brij-35 and incubated for 1 h at 37°C without or with one or more of the DUBs Otulin, AMSH-LP and USP2 (each at 1 µM) as indicated in the figure legends. The beads were washed twice with 50 mM Tris–HCl pH 7.5, 1% (v/v) Triton X-100, once with 50 mM Tris–HCl pH 7.5 and resuspended in 19 mM sodium carbonate, 22 mM sodium bicarbonate pH 9.0 and incubated for 1 h at 37°C with or without 0.5 M hydroxylamine. The beads were washed twice with 50 mM Tris–HCl pH 7.5, 1% (v/v) Triton X-100 and proteins eluted by resuspension for 10 min at 37°C in Lithium Dodecyl Sulfate sample buffer (Invitrogen) diluted four-fold in 2.5% (v/v) 2-mercaptoethanol. The eluted proteins were passed through a Spin-X centrifuge filter (Corning Costar) and subjected to SDS–PAGE using precast 4–12% gels and transferred to PVDF membranes prior to immunoblotting.

An amount of 1.0 mL of the modified collagen was dialyzed against water and freeze-dried. About 1 mg (by weight) was acid hydrolyzed, dried, and taken into 1.0 mL of water for filtration through Spin-x centrifuge filters (Corning Inc.). An amount of 100 μg collagen based on hydroxyproline content was aliquoted and spiked with an isotopically labeled standard mixture as described.^{13 (link)} This was dried in vacuo and reconstituted to 100 μL in buffer. An amount of 20 μL of this solution was used for analysis (or the equivalent of a 20 μg injection). Carboxymethyl-lysine (CML), carboxyethyl-lysine (CEL), and methylglyoxal-derived hydroimidazolone (MG-H1) were determined in acid hydrolysates of processed collagen samples by electron spray positive ionization-mass spectrometric multiple reaction monitoring (ESI_MRM) using LC-MS/MS system composed of a 2690 Separation module with a Quattro Ultima triple quadrupole mass spectrometry detector (Water-Micromass) following the procedure published by Ahmed and Thornalley.^{14 (link)} Equal amounts of collagen (20 μg), whereby collagen content was determined by a hydroxyproline colorimetric assay as described earlier (2), were injected for analysis. Pentosidine was assayed by HPLC as previously described.^{15 (link)} All results are expressed as nmol or pmol analyte per mg of collagen.