重组Anti-CD80抗体[EPR1157(2)] - BSA and Azide free (ab271905)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134120).

Flow cytometry overlay histogram showing wild-type Raji (green line) and CD80 knockout Raji stained with ab134120 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised 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 (ab134120) (1x 10⁶ in 100μl at 0.2 μg/ml (1/10150)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control was used at the same concentration and conditions as the primary antibody (wild-type Raji - black line, CD80 knockout Raji - 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 gave a positive signal in Raji Fixed with 80% methanol (5 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling CD80 with ab134120 at a dilution of 1/1000. Heat mediated antigen retrieval was performed using Trs/EDTA buffer, pH9 (ab93684). ImmunoHistoProbe one step HRP Polymer was used. Counter stained with hematoxylin.

The image shows cytoplasmic and membrane staining.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134120).

All lanes : Anti-CD80 antibody [EPR1157(2)] (ab134120) at 1/1000 dilution

Lane 1 : Wild-type Raji (Human Burkitt's lymphoma cell line) whole cell lysate
Lane 2 : CD80 knockout Raji (Human Burkitt's lymphoma cell line) whole cell lysate
Lane 3 : Ramos (Human Burkitt's lymphoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 33 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?

This data was developed using the same antibody clone in a different buffer formulation (ab134120).

Lanes 1 - 3: Merged signal (red and green). Green - ab134120 observed at 55 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

ab134120 was shown to react with CD80 in wild-type Raji cells in western blot with loss of signal observed in CD80 knockout sample. Wild-type and CD80 knockout Raji cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab134120 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.