Flow cytometric analysis of Raji (human Burkitt's lymphoma cell line) cell line labeling CD86 with ab239075 at 1/500 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

Gated on viable cells.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Daudi (human Burkitt's lymphoma cell line) cells labeling CD86 with ab239075 at 1/100 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous staining in Daudi cell line is observed.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

All lanes : Anti-CD86 antibody [EPR21962] (ab239075) at 1/1000 dilution

Lane 1 : Wild-type Raji cell lysate
Lane 2 : CD86 knockout Raji cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 38 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?

Lanes 1 - 2: Merged signal (red and green). Green - ab239075 observed at 70 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

ab239075 was shown to react with CD86 in wild-type Raji cells in western blot with loss of signal observed in CD86 knockout sample. Wild-type and CD86 knockout Raji cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab239075 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.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Raji (human Burkitt's lymphoma cell line) cells labeling CD86 with ab239075 at 1/100 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous staining in Raji cell line is observed.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

CD86 was immunoprecipitated from 0.35 mg of Raji whole (human Burkitt's lymphoma cell line) cell lysate with ab239075 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab239075 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1: Raji whole cell lysate 10 µg (Input). 

Lane 2: ab239075 IP in Raji whole lysate. 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab239075 in Raji whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 3 minutes.

All lanes : Anti-CD86 antibody [EPR21962] (ab239075) at 1/1000 dilution

Lane 1 : Ramos (human Burkitt's lymphoma cell line) whole cell lysate
Lane 2 : Raji (human Burkitt's lymphoma cell line) whole cell lysate
Lane 3 : Daudi (human Burkitt's lymphoma cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 38 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

Exposure time : Lane 1: 3 minutes; Lanes 2-3: 26 seconds.

Blocking/Dilution buffer: 5% NFDM/TBST.