Flow cytometry analysis of CD58 showing positive staining in the membrane of PBMC cells compared to an isotype control (blue). Human blood was collected, combined with a hydrophilic polysaccharide, centrifuged, transferred to a conical tube and washed with PBS. 50 ul of cell solution was added to each tube at a dilution of 2x10^7 cells/ml, followed by the addition of 50 ul of isotype control and ab171087 (0.5 ug/test). Cells were incubated for 30 min at 4ºC and washed with a cell buffer, followed by incubation with a DyLight 488-conjugated goat anti-mouse IgG (H+L) secondary for 30 min at 4ºC in the dark. FACS analysis was performed using 400 ul of cell buffer.

All lanes : Anti-CD58 antibody [TS2/9] (ab171087) at 1/250 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : CD58 knockout HAP1 whole cell lysate
Lane 3 : THP1 whole cell lysate
Lane 4 : Raji whole cell lysate

Lysates/proteins at 40 µg per lane.

Predicted band size: 24 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab171087 observed at 43 kDa. Red - loading control, ab176560, observed at 50 kDa.

ab171087 was shown to specifically react with CD58 in wild-type HAP1 cells as signal was lost in CD58 knockout cells. Wild-type and CD58 knockout samples were subjected to SDS-PAGE. Ab171087 and ab176560 (Rabbit anti alpha Tubulin loading control) were incubated overnight at 4°C at 1/250 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Immunofluorescent analysis of CD58 (green) showing staining in the cytoplasm of Raji cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a CD58 monoclonal antibody (ab171087) in 3% BSA-PBS at a dilution of 1:20 and incubated overnight at 4ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Flow cytometry analysis of CD58 showing positive staining in the membrane of PBMC cells. Human blood was collected, combined with a hydrophilic polysaccharide, centrifuged, transferred to a conical tube and washed with PBS. 50 ul of cell solution was added to each tube at a dilution of 2x10^7 cells/ml, followed by the addition of 50 ul of isotype control and ab171087 (0.5 ug/test). Cells were incubated for 30 min at 4ºC and washed with a cell buffer, followed by incubation with a DyLight 488-conjugated goat anti-mouse IgG (H+L) secondary for 30 min at 4ºC in the dark. FACS analysis was performed using 400 ul of cell buffer.

Flow cytometry analysis of CD58 showing weakly positive staining in the membrane of BAF-3 cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/ml, fixed with 2% paraformaldehyde, washed with PBS, and incubated with ab171087 (0.25 ug/test) for 60 min at room temperature. Cells were then blocked in a solution of 2% BSA-PBS for 30 min at room temperature, incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody, and re-suspended in PBS for FACS analysis.

All lanes : Anti-CD58 antibody [TS2/9] (ab171087) at 1/100 dilution

Lane 1 : Raji cell lysate
Lane 2 : Jurkat cell lysate
Lane 3 : BAF-3 cell lysate

Lysates/proteins at 25 µg per lane.

Predicted band size: 24 kDa
Observed band size: 24 kDa