Gel filtration protein standards

HeLa cell lines (RAB37 overexpression, RAB37 knockdown and control HeLa cells) and two transiently transfected cells (FLAG-RAB37-Q89L, FLAG-RAB37-T43N) were cultured in DMEM medium with 10% FBS and starved in the medium EBSS for 1 h. The cells were lysed and then centrifuged at 50,000g for 30 min. The supernatants (cytosol fraction) were filtered through 0.22 μm filter. Then the fractions were separated by size-exclusion chromatography on a Superose 6 increase 10/300 GL column (Cat# 29-0915-96, GE Healthcare Life Sciences, Fairfield, Connecticut, USA), which has a very broad fractionation range (Mr 5,000–5,000,000), especially for large proteins and complexes, and eluted at a flow rate of 0.5 ml min^–1 using the solution (50 mM sodium phosphate, 150 mM sodium chloride, pH 7.2). Fractions were subjected to Western blotting using relevant antibodies. The column was calibrated with gel filtration protein standards (Cat# 28-4038-42, GE Healthcare Life Sciences, Fairfield, Connecticut, USA) containing thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), Conalbumin (75 kDa), Ovalbumin (43 kDa).

Cy5-maleimide was purchased from GE Healthcare and AlexaFluor 488-maleimide from Life Technologies. Cation exchange chromatography column HiTrap SP HP, Gel filtration chromatography column Superdex 200 10/300 GL, and NAP5 as well as Gel Filtration protein standards were purchased from GE Healthcare. Amicon ultracentrifugation filter was purchased from Merck Millipore and Ni-NTA Agarose was from Qiagen. PVDF membrane was purchased from Roche Diagnostics and anti-strep tag antibody was from IBA.