All lanes : Anti-KPNB1 antibody [3E9] (ab2811) at 1/50 dilution

Lane 1 : U-251 MG (Human brain glioma cell line) whole cell lysate
Lane 2 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 3 : C6 (Rat glial tumor cell line) whole cell lysate

Lysates/proteins at 25 µg per lane.

Secondary
All lanes : HRP-conjugated secondary antibody

Predicted band size: 97 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

Immunofluorecent analysis of HMVEC (Human Lung Microvascular Endothelial cells) cells stained for KPNB1 using ab2811.

NIH/3T3 (Mouse embryo fibroblast cell line) cells were incubated for 4 minutes in PHEM/1%triton, washed for 2 minutes in 1x PHEM and fixed for 10 minutes at room temperature in 3.7% PFA containing 30mM sucrose. Following washing in PBS, the cells were incubated for 2 minutes in 100% Methanol at -20°C, then washed 3 times in PBS. The cells were then incubated with ab2811 (1/200) for 1 hour at room temperature. The image panel shows the nuclei stained with DAPI (blue) and the nuclear envalope and cytoplasm stained with ab2811 (green). 100x magnification.

 

 

Immunoprecipitation of Importin beta, in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells, using ab2811. Coomassie-stained 8% SDS-page gel was loaded with IP fractions obtained by incubating 2 mg of pre-cleared HeLa whole cell extracts with 4µg ab2811 or 4µg IgG (control). The Importin band (see arrow) was cut out of the gel and its identity confirmed by Mass Spectometry. Please refer to protocol tab for further experimental details.

Flow cytometry analysis of KPNB1 in Jurkat (Human T cell leukemia cell line from peripheral blood) cells (green) compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/ml, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with ab2811 (1ug/test) for 40 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated secondary antibody and re-suspended in PBS for FACS analysis.

All lanes : Anti-KPNB1 antibody [3E9] (ab2811) at 1/2000 dilution

Lane 1 : Mouse lung whole tissue lysate
Lane 2 : Mouse kidney whole tissue lysate
Lane 3 : Mouse spleen whole tissue lysate

Secondary
All lanes : HRP-conjugated Goat anti-mouse IgG polyclonal at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 97 kDa
Observed band size: 97 kDa


Exposure time: 3 minutes

See Abreview

Anti-KPNB1 antibody [3E9] (ab2811) at 1/5000 dilution + HeLa Whole cell extract

Secondary
HRP-conjugated anti mouse IgG at 1/5000 dilution

Performed under reducing conditions.

Predicted band size: 97 kDa



HeLa whole cell extract was run on a 10%SDS-PAGE and transferred to PVDF membrane. Membrane was blocked for 30 mins in TBS/0.1% Tween/ 5% Milk; ab2811 (1/5000) was incubated for 1 hr in TBS/0.1%Tween/5% Milk and followed by 3 washes in TBS/ 0.1%Tween (3x 7 mins). Secondary antibody was incubated for 30 mins in a TBS/ 0.1% Tween solution.This was followed by 3 washes with the TBST solution (3x7 mins) and one wash in TBS. Western blot developed using ECL plus (Amersham).

Immunolocalization of KPNB1 in PTK (Long-nosed potoroo epithelial kidney cell line) cells using ab2811.

Flow cytometry analysis of KPNB1 in PC-12 (Rat adrenal gland pheochromocytoma cell line) cells (green) compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/ml, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with ab2811 (1ug/test) for 40 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated secondary antibody and re-suspended in PBS for FACS analysis.

Flow cytometry analysis of KPNB1 in 3T3 cells (green) compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/ml, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with ab2811 (1ug/test) for 40 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated secondary antibody and re-suspended in PBS for FACS analysis.

Overlay histogram showing Jurkat cells stained with ab2811 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab2811, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Jurkat cells fixed with methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.