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Perfusion-biotinylation of IMCD apical membrane proteins

A rat kidney inner medulla (orange box, Fig. 1A) was excised and placed on a porous support that allows fluid removal and in between filter papers that moisturize the tissue (Fig. 1B). Apical membrane proteins were labeled with biotin via perfusing the IMCD lumens using a custom-made double barreled pipette in the cold room (2 ºC) to inhibit endocytosis. One barrel delivered paraformaldehyde to fix the membrane lipids before the other barrel delivered sulfo-NHS-LC-biotin or sulfo-NHS-SS-biotin to label the apical membrane proteins. The fixation of lipids prior to protein biotinylation was necessary to ensure apical protein biotinylation. Blue and red food dyes were used in the perfusates to visualize fluid changes in the IMCDs. A second pipette above the perfusion pipette applied Tris buffer to moisturize the tissue and to quench the biotinylation reagent. Figure 1C shows the perfusion setup in the cold room.

Fig. 1A	Fig. 1B	Fig. 1C
A rat kidney	An isolated inner medulla under perfusion biotinylation	A picture of experimental settings

Restricted apical protein biotinylation after fixation of membrane lipids

Perfusion-biotinylation of the IMCDs without fixation resulted in intracellular biotinylation as revealed by FITC-streptavidin that stains biotin in green (Fig. 2A). The biotinylation was nevertheless confined to the perfused IMCDs as indicated by IMCD marker protein AQP2 staining (Fig. 2A, red). Nuclei stained with DAPI appear blue. Bar = 10 mM. The labeled proteins and their associated proteins from the non-fixed perfusion-biotinylated IMCDs (Technique 10c) were isolated with streptavidin-agarose beads and prepared for LC-MS/MS protein identification (LTQ).

Perfusion-biotinylation of IMCDs after paraformaldehyde fixation prevented intracellular biotinylation, resulting in linear labeling of the apical surface (Fig. 3B, green). AQP2 and DAPI nuclear staining are red and blue, respectively. The labeled proteins and their associated proteins (from the fixed perfusion-biotinylated IMCDs Technique 10a) were isolated with CaptAvidin-agarose beads and prepared for LC-MS/MS protein identification (LTQ).

Fig. 2A	Fig. 2B
Cross section of non-fixed perfusion-biotinylated IMCD	Cross section of fixed perfusion-biotinylated IMCD