Anti-CD58 antibody [TS2/9]

6 product images

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  Flow Cytometry - Anti-CD58 antibody [TS2/9] (ab171087)

  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 4°C 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 4°C in the dark. FACS analysis was performed using 400 ul of cell buffer.

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  Flow Cytometry - Anti-CD58 antibody [TS2/9] (ab171087)

  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 4°C 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 4°C in the dark. FACS analysis was performed using 400 ul of cell buffer.

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  Western blot - Anti-CD58 antibody [TS2/9] (ab171087)

  Lanes 1 - 4: Merged signal (red and green). Green - ab171087 observed at 43 kDa. Red - loading control, Anti-alpha Tubulin antibody [EPR13478(B)] - 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 Anti-alpha Tubulin antibody [EPR13478(B)] - Loading Control 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 Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

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

  Lane 1: Wild-type HAP1 whole cell lysate at 40 µg

  Lane 2: CD58 knockout HAP1 whole cell lysate at 40 µg

  Lane 3: THP1 whole cell lysate at 40 µg

  Lane 4: Raji whole cell lysate at 40 µg

  Predicted band size: 28 kDa

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  Flow Cytometry - Anti-CD58 antibody [TS2/9] (ab171087)

  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.

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  Immunocytochemistry/ Immunofluorescence - Anti-CD58 antibody [TS2/9] (ab171087)

  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 4°C 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.

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  Western blot - Anti-CD58 antibody [TS2/9] (ab171087)

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

  Lane 1: Raji cell lysate at 25 µg

  Lane 2: Jurkat cell lysate at 25 µg

  Lane 3: BAF-3 cell lysate at 25 µg

  Predicted band size: 28 kDa

  Observed band size: 24 kDa