ab128797 staining CD11b in Raw264.7 (LPS treated) cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Tween 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 ab128797 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. 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 ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

Overlay histogram showing RAW 264.7 cells stained with ab128797 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab128797, 0.1μg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (polyclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

CD11b was immunoprecipitated using 0.5mg RAW 264.7 whole cell extract, 5µg of Rabbit polyclonal to CD11b and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).

The antibody was incubated under agitation with Protein G beads for 10min, RAW 264.7 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab128797.

Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).

Band: 170kDa, non specific bands - 42, 55 and 65kDa: We are unsure as to the identity of this extra band; CD11b

Anti-CD11b antibody (ab128797) at 1 µg/ml + RAW 264.7 (Mouse leukaemic monocyte macrophage cell line) Whole Cell Lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 127 kDa
Observed band size: 170 kDa why is the actual band size different from the predicted?
Additional bands at: 42 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 4 minutes


CD11b contains a number of potential glycosylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted. The predicted molecular weight of CD11b is 127 kDa (SwissProt), however we expect to observe a banding pattern around 170 kDa. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.