All lanes : Anti-CD74 antibody [LN2] (ab9514) at 5 µg/ml

Lane 1 : Wild-type Raji cell lysate
Lane 2 : CD74 knockout Raji cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : HepG2 cell lysate

Lysates/proteins at 30 µg per lane.

Performed under reducing conditions.

Predicted band size: 34 kDa
Observed band size: 35 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab9514 observed at 35 kDa. Red - loading control, ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) observed at 55 kDa.

ab9514 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 fluorescent western blot (TBS-based) blocking solution before incubation with ab9514 and ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) overnight at 4 °C at 5 µg/ml and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

Ab9514 staining CD74 in paraffin embedded Human tonsil tissue sections by Immunohistochemistry (IHC-P).

Flow cytometry overlay histogram showing wild-type Raji (green line) and CD74 knockout Raji cells (ab273378) stained with ab9514 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then 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 (ab9514) (1x10⁶ in 100μl at 1 μg/ml) for 30 min at 22°C.

The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor^® 488, pre-adsorbed) (ab150117) was used at 1/2000 for 30 min at 22°C.

Isotype control antibody was mouse IgG1κ (ab170190) 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.

This antibody can also be used in Raji cells fixed with 4% formaldehyde (10 min) / permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions.

 

Overlay histogram showing Raji cells stained with ab9514 (red line). The cells were fixed with 80% methanol (5 min) and incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab9514, 1/10 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.
Please note that Abcam do not have any data for use of this antibody on non-fixed cells. We welcome any customer feedback.

Anti-CD74 antibody [LN2] (ab9514) at 5 µg/ml + Human tonsil normal tissue lysate - total protein (ab29615) at 10 µg

Secondary
Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 34 kDa
Observed band size: 34 kDa
Additional bands at: 37 kDa (possible post-translational modification)

Ab9514 staining CD74 in paraffin embedded Human tonsil tissue sections by Immunohistochemistry (IHC-P).

ab9514 staining CD74 in human liver tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with neutral buffered formalin and a heat mediated antigen retrieval step was performed using EDTA buffer pH 9.0. Samples were then blocked with 10% serum for 10 minutes at 20°C followed by incubation with the primary antibody at a 1/75 dilution for 30 minutes at 20°C. A HRP-conjugated rat anti-mouse/rabbit polyclonal was used undiluted as the secondary antibody.

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