Pulldowns 

In a final volume of 30 μL, 20 μg of purified MBP-Rubicon RH domain was mixed with 20 μg of RAB7A in 50 mM HEPES 7.5, 150 mM NaCl, 2 mM MgCl2, 10 mM TCEP buffer.

Include a negative control consisting of only soluble Rab7, with no MBP-Rubicon, in order to test your washing efficacy.

Incubate samples on rocker at room temperature for 1 H

Add 20 uL of 50% v/v amylose resin to each sample

Allow to rock for an additional 30 min to bind

Collect resin at bottom of tube via a tabletop centrifuge, aspirate off and discard supernatant, and wash with 500 uL of ice cold buffer for three total washes.

Elute sample by resuspending beads in 20 uL of buffer + 20 mM maltose for 10 min on shaker.

Prepare samples for SDS-PAGE by adding 1x loading buffer, and load onto a 4-12% gel. Run gel at 120 V until dye front reaches the bottom of the gel

Stain with Coomassie blue G-250, and destain with water until bands are clearly visible. Image.