Analysis of co-localisation of FcRn with cellular markers.

HepG2 (Human liver hepatocellular carcinoma cell line) cells were fixed, permeabilized and co-stained with anti-FcRn humanised Fab’, followed by anti-human IgG conjugated Alexa Fluor^®488 (Top left panel. Bar 5 μm) and ab84036 followed by anti-rabbit/mouse conjugated Alexa Fluor^®568 (Top right panel). FcRn fluorescence is shown in green and marker fluorescence is shown in red, yellow fluorescence in the overlay images (Bottom left panel) indicates co-localization.

Images are magnifications of the white boxed area (Bottom right panel. Bar 20 μm). 

All lanes : Anti-Transferrin Receptor antibody (ab84036) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) whole cell lysate
Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) whole cell lysate
Lane 3 : U-2 OS (Human osteosarcoma cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : 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: 84 kDa
Observed band size: 98 kDa why is the actual band size different from the predicted?
Additional bands at: 37 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 4 minutes


Transferrin Receptor contains a number of potential glycosylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted.

ICC/IF image of ab84036 stained HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

The cells were fixed in 4% formaldehyde (10 minutes) then permeabilized using 0.1% PBS-Triton and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour to further permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab84036 at 1 µg/ml overnight at +4°C.

The secondary antibody (pseudo-colored green) was an Alexa-Fluor^® 488 goat anti- rabbit (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1 hour. Alexa-Fluor^® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1 hour at room temperature.

DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43 µM for 1 hour at room temperature.

Anti-Transferrin Receptor antibody (ab84036) at 1 µg/ml + Mouse spleen tissue 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: 84 kDa
Observed band size: 100,50 kDa why is the actual band size different from the predicted?


Exposure time: 8 minutes


Transferrin Receptor contains a number of potential glycosylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted.

Overlay histogram showing MCF7 cells stained with ab84036 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab84036) (1x10⁶ in 100µl at 1 µg/ml) for 30 min at 22ºC.

The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor® 488, pre-adsorbed) (ab150117) was used at 1/2000 for 30 min at 22ºC.

Isotype control antibody (black line) was Rabbit IgG (polyclonal) (ab171870) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.

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