Anti-Lamin B1 antibody - Nuclear Envelope Marker(AB16048)

All lanes:

Western blot - Anti-Lamin B1 antibody - Nuclear Envelope Marker (ab16048) at 1/1000 dilution

All lanes:

Human Pancreatic cell line - whole cell lysate at 20 µg

Secondary

All lanes:

HRP conjugated goat anti-rabbit antibody at 1/2000 dilution

Predicted band size: 66 kDa

Observed band size: 68 kDa

Developed using the ECL technique.

Exposure time: 30s

This image is courtesy of an anonymous Abreview

Immunohistochemical analysis of formalin-fixed paraffin-embedded human liver labelling lamin B1 with ab16048 at a concentration of 0.2µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32min with ULTRA cell conditioning solution (CC1 pH8.5). ab16048 anti lamin B1 antibody was incubated at 37°C for 16min. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

ab16048 staining Lamin B1 in HepG2 cells. The cells were fixed with 4% paraformaldehyde (10 min) 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 ab16048 at 0.1µg/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 ab150120 Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594) pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 100% methanol (5 min).

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

Human and mouse cells stained with ab16048 (1/500 dilution). The cells were fixed and permeabilized in 4% formaldehyde 0.2% Tritonm X100 for 10 minutes at room temperature then washed 3x in PBS.

A : HeLa cells + ab16048 (green)

B : HeLa cells counterstained with DAPI (blue)

C : 3T3 cells + ab16048 (green)

D : 3T3 cells counterstained with DAPI (blue)

ab16048 staining Lamin B1 in human infantile fibromatosis tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 1% FBS/BSA for 3 hours at room temperature; antigen retrieval was by heat mediation in Tris pH9. Samples were incubated with primary antibody (1/100 in TBS + 1% BSA + 1% FBS) for 16 hours. An undiluted HRP-conjugated goat anti-rabbit IgG polyclonal was used as the secondary antibody.

Human and mouse cells stained with ab16048 (1/500 dilution). The cells were fixed in 100% methanol for 6 minutes at -20°C then washed once in PBS.

A : HeLa cells + ab16048 (green)

B : HeLa cells counterstained with DAPI (blue)

C : 3T3 cells + ab16048 (green)

D : 3T3 cells counterstained with DAPI (blue)

IHC image of Lamin B1 staining in Human normal Liver formalin fixed paraffin embedded tissue section^* performed on a Leica Bond^™ 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 20 mins. The section was then incubated with ab16048 1μg/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.

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

Immunocytochemistry analysis of formaldehyde-fixed NP40-permeabilized Mouse vascular smooth muscle cells staining with ab16048 at 1/100 dilution. Secondary antibody was Alexa Fluor^® 488 donkey Anti-rabbit at 1/500 dilution. Samples were incubated with the primary antibody with 3% BSA in PBS for 12 hours at 4°C. Blocking was done using 3% BSA for 1 hour at 21°C.

This image is courtesy of a customer review

Lane 1 : Wild-type HAP1 nuclear lysate (10 μg)

Lane 2 : Lamin B1 knockout HAP1 nuclear lysate (10 μg)

Lanes 1 and 2 : Green signal from target - ab16048 observed at 68 kDa. Red signal from loading control - ab10799 observed at 18 kDa.

ab16048 was shown to specifically react with lamin B1 in wild-type HAP1 cells. No band was observed knockout samples were used. Wild-type and lamin B1 knockout samples were subjected to SDS-PAGE. ab16048 and ab10799 (loading control to histone H3 at 0.1μg/mL) were both incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-Lamin B1 antibody - Nuclear Envelope Marker (ab16048) at 1 µg/mL

Lane 1:

Wild-type HAP1 nuclear lysate at 10 µg

Lane 2:

Lamin B1 knockout HAP1 nuclear lysate at 10 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 36 kDa,66 kDa

Observed band size: 68 kDa

Blocking buffer : 3% Milk

Gel type : MOPS

Exposure Time : 2 minutes

Observed band size : 73 kDa

Additional bands : 46 kDa

All lanes:

Western blot - Anti-Lamin B1 antibody - Nuclear Envelope Marker (ab16048) at 1 µg/mL

Lane 1:

HeLa whole cell lysate at 10 µg

Lane 2:

PC12 whole cell lysate at 10 µg

Lane 3:

NIH 3T3 whole cell lysate at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/50000 dilution

Predicted band size: 66 kDa

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)

Lane 2 : empty lane

Lane 3 : KO HAP1 LMNB1 whole cell lysate (20 μg)

Lane 4 : empty lane

Lanes 1 - 4 : Merged signal (red and green). Green - ab16048 observed at 70 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab16048 was shown to specifically react with LMNB1 (Lamin B1) in wild type HAP1 cells. No band was observed when LMNB1 (Lamin B1) knockout samples were used. ab16048 LMNB1 (Lamin B1) and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 0.1 μg per mL and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Lamin B1 antibody - Nuclear Envelope Marker (ab16048)

Predicted band size: 66 kDa

All lanes:

Western blot - Anti-Lamin B1 antibody - Nuclear Envelope Marker (ab16048) at 1/1000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg with Mouse Lamin B1 peptide (ab16375)

Secondary

All lanes:

Alexa fluor goat polyclonal to Rabbit IgG at 1/10000 dilution

Predicted band size: 66 kDa

Observed band size: 68-70 kDa