Rabbit anti rab7

Late phagosomes were purified from J774E cells 10 min/20 min (pulse/chase) after phagocytosis of 2 μm latex beads as described for LBPs containing 1 μm latex beads above. Purified LBPs were incubated for 30 min at 37°C with 10 μM hexahistidine-tagged Arl8b(Q75L) or Arl8b(T34N), in HB containing an ATP-regenerating system, 1x salts, and 1 mM DTT.^{33 (link)} LBPs were isolated from reaction mixtures by density gradient centrifugation and fixed onto glass coverslips. Samples were sequentially incubated with PBS/50 mM NH₄Cl and with blocking buffer (PBS/4% goat serum) for each 15 min at ambient temperature. LBPs were incubated with a mouse anti-Histidine tag antibody (1:50 in blocking buffer) for 60 min, washed five times with PBS, and incubated with an Alexa 535-conjugated goat anti-mouse antibody (1:200, blocking buffer). Samples were washed five times with PBS and incubated with rabbit anti-Rab2 or rabbit anti-Rab7 (Abcam) antibodies (each 1:50, blocking buffer) for 60 min at ambient temperature. Samples were washed five times with PBS and incubated with an Alexa 488-labeled goat anti-rabbit antibody (30 min, 1:200 in blocking buffer). Samples were washed four times with PBS, twice with ddH₂O and mounted in Mowiol. Samples were analyzed using a Zeiss Axio Imager equipped with Apotome and a 100x oil immersion lens.