PRODUCT-SPECIFIC PROTOCOLS

FC (UBE2D1/2/3/4)
Sample type:	A431 cell, RAW2647 cell, RH35 cell
Sample preparation:	4% paraformaldehyde and permeabilization buffer
Blocking buffer:	normal goat serum
Primary antibody concentration:	1μg/1x10⁶ cells
Incubation time:	overnight at 4°C

PROTOCOL

Centrifugation: centrifuge prepared single cell suspension at 3000 rpm for 2 minutes and aspirate supernatant to collect cells.
Fixation: resuspend cells in 1 ml of 4% Paraformaldehyde. Transfer 4% Paraformaldehyde containing cells into a 1.5 ml centrifuge tube. Fix at 4°C for 20 minutes.
Washing: centrifuge the cells at 3000 rpm for 2 minutes. Discard supernatant in appropriate waste container. Resuspend and wash cells in 1 ml DPBS. Repeat the washing step twice.
Prepare tubes for samples and indicate sample type, control and antibody information on the tubes.
Permeabilization: permeabilize cells by adding permeabilization buffer to cells. The volume of the permeabilization buffer is 100μl x number of tubes for samples, which is adding 100μl of permeabilization buffer to each tube (1x10⁶ cells per tube).

Do not add any antibody into the Blank tube. Aliquot 100μl of the cell suspension into each sample tube. Add 5μl of concentrated goat serum into every other tube and mix thoroughly. Incubate at 4°C for 10 minutes.

Add 1μg of rabbit IgG to the Isotype control tube. Add 1μg of primary antibody (1μg/1x10⁶ cells) into each tube. Mix well and incubate at 20°C for 30 minutes.
Centrifuge at 3000 rpm for 2 minutes and discard the supernatant.
Resuspend cells in 1 ml DPBS twice and add 100μl DPBS.

Do not add any antibody into the Blank tube. Add 5μl DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10μg/1x10⁶ cells) into every other tube. Mix well and incubate at 20°C for 30 minutes, avoiding direct light.
Centrifuge at 3000 rpm for 2 minutes and discard the supernatant.
Resuspend cells in 1 ml DPBS twice and add 100μl DPBS, mix well.