Cells: HT29-N2 Tspan8.GFP (clone 3)
Treatments: 4E6 cells were trasnfected at the moment of plating (60mm dish) with Stx2.FLAG.
IP: Two days after transfection, cells were lysed with lysis buffer not containing EDTA and DTT. samples were maintain in a rotating weel for 15 minutes at 4C and cetrifugated at 12000g for 15 min at 3C.

antiVamp8 (abcam - Rbb) was coupled to Dynabeads_ProteinG by incutagin 20ug of antibodies with 200ul of dyanbeads in PBST (tween 0.001%) ~45minutes at RT in a rotating wheel. As control, were not incubated with any antibody.

The CoIP was done for 60 minutes at 4C in a rotating wheel.

Samples were run in a 12% SDS gel in this order: Control, Vamp8-coated dynabeads. First 2 are 20ul of the inputs and last 2 are all the CoIP samples ~35ul.

Transfer was done ON at 4C and 40mA into a PVDF membrane.

The membrane was cut and incubated ON at 4C with anti GFP, anti Stx2 and antiVamp8 antibodies, each one of them diluted in TBST/BSA2%.

The region around 25KDa was cut off the incubation to remove the small IgG of the secondary antibody incubation as they are of the same species.

The names of the folders indicate the primary antibody used. For some antibodies, several exposure times were acquired.

A pdf file of the protein ladder used is included for reference of the molecular weigths.
