Back to "Techniques Used"

Incubation-biotinylation of IMCD basement membrane and basolateral membrane proteins

IMCDs were separated from non-IMCDs of whole inner medullas via enzymatic digestion and low speed centrifugation. The isolated IMCD fraction (lane I, Fig. A) was enriched with IMCD marker protein AQP2 and depleted of non-IMCD marker protein AQP1, compared to whole inner medulla (lane W, Fig. A) and non-IMCD homogenates (lane N, Fig. A). These isolated IMCDs were labeled with biotin via incubation with sulfo-NHS-SS-biotin or sulfo-NHS-LC-biotin on ice. Without (Fig. B) or with (Fig. C) fixation prior to biotinylation, the labeling occurred at the basement membrane and the basolateral membranes of the IMCDs as revealed by confocal fluorescent microscopy using streptavidin–FITC to stain sites of biotinylation (green, Fig. B and C). AQP2 and nuclear DAPI staining are shown in red and blue. Bar = 5 mm. Biotinylated proteins and their associated proteins in the non-fixed (Technique10c) and the fixed (Technique 10b) incubation-biotinylated IMCDs were isolated with streptavidin-agarose beads and prepared for LC-MS/MS (LTQ) protein identification.

Fig. A	Fig. B	Fig. C
AQP1 and AQP2 immunoblot of whole inner medulla, non-IMCD, and IMCD homogenates	Non-fixed incubation-biotinylated IMCD cross section	Fixed incubation-biotinylated IMCD cross section