All lanes : Anti-Lamin B Receptor/LBR antibody [BBmLBR 12.F8] (ab232731) at 1 µg/ml

Lane 1 : Wild-type HEK-293 whole cell lysate
Lane 2 : Human LBR (Lamin B Receptor) knockout HEK-293T cell lysate (ab257503)
Lane 3 : Wild-type MEF-1 whole cell lysate
Lane 4 : LBR knockout MEF-1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) at 1/20000 dilution

Performed under reducing conditions.

Predicted band size: 71 kDa
Observed band size: 58 kDa

why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab232731 observed at 58 kDa. Red - loading control, ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) observed at 37kDa.

ab232731 was shown to react with Lamin B Receptor (LBR) in wild-type HEK-293 and MEF-1 cells in western blot. Loss of signal was observed when LBR knockout samples were used. Wild-type and LBR knockout (ab257503) HEK-293 cell lysates  and wild-type and LBR knockout MEF-1 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk before incubation with ab232731 and ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) overnight at 4°C at 1 µg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

LBR knockout MEF-1 samples were kindly provided by the Brian Burke laboratory, A-Star Institute, Singapore.

IHC image of Lamin B receptor staining in a section of formalin-fixed paraffin-embedded normal human duodenum* performed on a Leica Bond^TM system using the standard Protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab232731, 0.05µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

ab232731 staining Lamin B Receptor in HEK-293 cells. The cells were fixed with 4% paraformaldehyde (10min), permeabilized with 0.1%PBS-Triton X-100 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 ab232731 at 10µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control, at 1/1000 dilution. Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) 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 pseudocolor red). Nuclear DNA was labeled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

All lanes : Anti-Histone H4 antibody [EPR16599] - BSA and Azide free (ab232371) at 1 µg/ml

Lane 1 : MEF-1 wild-type whole cell lysate
Lane 2 : MEF-1 LBR knockout whole cell lysate

Lysates/proteins at 10 µg per lane.

Performed under reducing conditions.

Predicted band size: 71 kDa
Observed band size: 65 kDa why is the actual band size different from the predicted?

This blot was produced using a 4-12% Bis-tris under the MOPS buffer system. The gel was run at 200V for 55 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was blocked for an hour using 3% milk before ab232731 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at a 1ug/ml concentration and 1/20000 dilution respectively. Antibody binding was detected using Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Cell samples kindly provided by the Brian Burke laboratory, A-Star Institute, Singapore.