Flow Cytometry analysis of Ramos cells labelling CD27 with purified ab131254 at 1/300 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

All lanes : Anti-CD27 antibody [EPR8569] (ab131254) at 1/1000 dilution

Lane 1 : Wild-type Raji cell lysate
Lane 2 : CD27 knockout Raji cell lysate
Lane 3 : Ramos cell lysate
Lane 4 : Human brain tissue lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 29 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 - ab131254 observed at 35 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

ab131254 was shown to react with CD27 in wild-type Raji cells in western blot with loss of signal observed in CD27 knockout sample. Wild-type and CD27 knockout Raji cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab131254 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at a 1 in 1000 dilution 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 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling CD27 with purified ab131254 at 1/1500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500).

Negative control using PBS instead of primary antibody.

Counterstained with hematoxylin.

Anti-CD27 antibody [EPR8569] (ab131254) at 1/5000 dilution (purified) + NAMALWA (human Burkitt's lymphoma cell line) cell lysate at 20 µg

Secondary
Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 29 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes : Anti-CD27 antibody [EPR8569] (ab131254) at 1/1000 dilution (purified)

Lane 1 : Human lymph node tissue lysate
Lane 2 : Human fetal spleen tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 29 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung carcinoma tissue showing negative staining with purified ab131254 at 1/800.

Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500).

Negative control using PBS instead of primary antibody.

Counterstained with hematoxylin.

Immunocytochemistry/Immunofluorescence analysis of Jurkat cells labelling CD27 with purified ab131254 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) were also used.

Control 1: primary antibody (1/500) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

All lanes : Anti-CD27 antibody [EPR8569] (ab131254) at 1/1000 dilution (unpurified)

Lane 1 : Ramos (human Burkitt's lymphoma cell line) cell lysate
Lane 2 : Human lymph node cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled Goat anti Rabbit IgG at 1/2000 dilution

Predicted band size: 29 kDa

Immunohistochemistry (Formalin/PFA-fixed paraffin embedded sections) analysis of human stomach tissue labelling CD27 with ab131254 at a dilution of 1/100.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin embedded sections) analysis of human tonsil tissue labelling CD27 with ab131254 at a dilution of 1/100.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Equilibrium disassociation constant (K_D)
Learn more about K_D

Click here to learn more about K_D