Anti-CD27 antibody [EPR8569] - BSA and Azide free

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CD27 antibody [EPR8569] - BSA and Azide free (AB256583)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab131254).

Flow cytometry staining of human peripheral blood mononuclear cells (PBMCs) with ab131254 (right) or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (left). Cells were fixed and permeabilised with BD Cytofix/Cytoperm™ for 20 min. PBMCs were incubated for 30 min at 22°C in 1x PBS containing 10 μg/ml human IgG and 10 % normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody ab131254 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (1x 106 in 100 μl at 0.04 μg/ml (1/52750)) for 30 min at 4°C . The cells were simultaneously stained with CD4.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 4°C

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on viable cells.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CD27 antibody [EPR8569] - BSA and Azide free (AB256583)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab131254).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD27 antibody [EPR8569] - BSA and Azide free (AB256583)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab131254).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD27 antibody [EPR8569] - BSA and Azide free (AB256583)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab131254).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CD27 antibody [EPR8569] - BSA and Azide free (AB256583)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab131254).

ab131254 staining CD27 in Raji cells, with negative expression in A375 cells. The cells were fixed with 100% methanol (5 min), permeabilised with 0.1% Triton x-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab131254 at 1 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 0.5 μg/ml. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150119, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 647), pre-adsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

This product also work with 4% formaldehyde (10 min) fixation under the same testing conditions.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD27 antibody [EPR8569] - BSA and Azide free (AB256583)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab131254).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD27 antibody [EPR8569] - BSA and Azide free (AB256583)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab131254). Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human skeletal muscle showing negative staining with purified ab131254 at 1/1800. Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ab214880, a Goat Anti-Rabbit IgG H&L (HRP polymer) was used as the secondary antibody (1/500). Counterstained with hematoxylin. Negative control : No staining on human skeletal muscle. The section was incubated with ab131254 at 4°C overnight.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CD27 antibody [EPR8569] - BSA and Azide free (AB256583)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab131254).

• WB

Lab

Western blot - Anti-CD27 antibody [EPR8569] - BSA and Azide free (AB256583)

This data was developed using the same antibody clone in a different buffer formulation (ab131254).

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.

All lanes:

Western blot - Anti-CD27 antibody [EPR8569] (<a href='/en-us/products/primary-antibodies/cd27-antibody-epr8569-ab131254'>ab131254</a>) at 1/1000 dilution

Lane 1:

Wild-type Raji (Human Burkitt's lymphoma cell line) whole cell lysate at 20 µg

Lane 2:

CD27 knockout Raji (Human Burkitt's lymphoma cell line) whole cell lysate at 20 µg

Lane 2:

Western blot - Human CD27A knockout Raji cell line (<a href='/en-us/products/cell-lines/human-cd27a-knockout-raji-cell-line-ab274910'>ab274910</a>)

Lane 3:

Ramos (Human Burkitt's lymphoma cell line) whole cell lysate at 20 µg

Lane 4:

Human brain tissue lysate at 20 µg

Predicted band size: 29 kDa

Observed band size: 35 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-CD27 antibody [EPR8569] - BSA and Azide free (AB256583)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.