Anti-Lamin B Receptor/LBR antibody [BBmLBR 12.F8] - BSA and Azide free

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Lamin B Receptor/LBR antibody [BBmLBR 12.F8] - BSA and Azide free (AB269576)

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).

This image was produced using the same antibody clone but in a different formulation ab232731, PBS and sodium azide.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lamin B Receptor/LBR antibody [BBmLBR 12.F8] - BSA and Azide free (AB269576)

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.

This image was produced using the same antibody clone but in a different formulation ab232731, PBS and sodium azide.

• WB

Lab

Western blot - Anti-Lamin B Receptor/LBR antibody [BBmLBR 12.F8] - BSA and Azide free (AB269576)

This data was developed using the same antibody clone in a different buffer formulation (ab232731).

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 HEK-293 cell lysates (ab257503) 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.

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

All lanes:

Western blot - Anti-Lamin B Receptor/LBR antibody [BBmLBR 12.F8] (<a href='/en-us/products/primary-antibodies/lamin-b-receptor-lbr-antibody-bbmlbr-12f8-ab232731'>ab232731</a>) at 1 µg/mL

Lane 1:

Wild-type HEK-293 whole cell lysate at 20 µg

Lane 2:

Western blot - Human LBR (Lamin B Receptor) knockout HEK-293T cell lysate (<a href='/en-us/products/cell-lysates/human-lbr-lamin-b-receptor-knockout-hek-293t-cell-lysate-ab257503'>ab257503</a>) at 20 µg

Lane 3:

Wild-type MEF-1 whole cell lysate at 20 µg

Lane 4:

LBR knockout MEF-1 whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-800cw-preadsorbed-ab216772'>ab216772</a>) at 1/20000 dilution

Predicted band size: 71 kDa

Observed band size: 58 kDa

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• WB

Lab

Western blot - Anti-Lamin B Receptor/LBR antibody [BBmLBR 12.F8] - BSA and Azide free (AB269576)

This image was produced using the same antibody clone but in a different formulation ab232731, PBS and sodium azide.

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.

All lanes:

Western blot - Anti-Lamin B Receptor/LBR antibody [BBmLBR 12.F8] (<a href='/en-us/products/primary-antibodies/lamin-b-receptor-lbr-antibody-bbmlbr-12f8-ab232731'>ab232731</a>) at 1 µg/mL

Lane 1:

MEF-1 wild-type whole cell lysate at 10 µg

Lane 2:

MEF-1 LBR knockout whole cell lysate at 10 µg

Predicted band size: 71 kDa

Observed band size: 65 kDa

false