Anti-CD74 antibody [CLIP/3127R]

• Flow Cyt

Lab

Flow Cytometry - Anti-CD74 antibody [CLIP/3127R] (AB270265)

Flow cytometry overlay histogram showing wild-type Raji (green line) and CD74 knockout Raji cells (ab273378) stained with ab270265 (red line). The cells were incubated in 1x PBS containing 10μg/ml human IgG and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab270265) (1x10⁶ in 100μl at 1 μg/ml) for 30 min at 4°C.

The secondary antibody Goat anti-rabbit IgG H&L (Alexa Fluor^® 488, pre-adsorbed) (ab150081) was used at 1/2000 for 30 min at 4°C.

Isotype control antibody was Rabbit IgG (monoclonal) (ab172730) used at the same concentration and conditions as the primary antibody (wild-type Raji cells - black line; CD74 knockout Raji cells ab273378 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• WB

Supplier Data

Western blot - Anti-CD74 antibody [CLIP/3127R] (AB270265)

All lanes:

Western blot - Anti-CD74 antibody [CLIP/3127R] (ab270265) at 2 µg/mL

All lanes:

HEK-293T cell lysate

Predicted band size: 34 kDa

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• Flow Cyt

Supplier Data

Flow Cytometry - Anti-CD74 antibody [CLIP/3127R] (AB270265)

Flow cytometric analysis of Raji cells labeling CD74 using ab270265 at 2 μg/10⁶ cells (Blue), followed by goat anti-mouse IgG-CF488; isotype control (Red).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD74 antibody [CLIP/3127R] (AB270265)

Formalin-fixed, paraffin-embedded human tonsil stained for CD74 using ab270265 at 2 μg/ml in imunnohistochemical analysis.

• WB

Lab

Western blot - Anti-CD74 antibody [CLIP/3127R] (AB270265)

Lanes 1 - 4 : Merged signal (red and green). Green - ab270265 observed at 35 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab270265 was shown to react with CD74 in western blot. The band observed in CD74 knockout cell line ab273378 (knockout lysate ab275529) below 35 kDa is likely to represent a truncated form. This has not been investigated further. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab270265 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at 1 μg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-CD74 antibody [CLIP/3127R] (ab270265) at 1 µg/mL

Lane 1:

Wild-type Raji cell lysate at 30 µg

Lane 2:

CD74 knockout Raji cell lysate at 30 µg

Lane 2:

Western blot - Human CD74 knockout Raji cell line (<a href='/en-us/products/cell-lines/human-cd74-knockout-raji-cell-line-ab273378'>ab273378</a>)

Lane 3:

Jurkat cell lysate at 30 µg

Lane 4:

HepG2 cell lysate at 30 µg

Predicted band size: 34 kDa

Observed band size: 35 kDa

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