Recombinant Anti-Lamin B1 antibody [EPR8985(B)] - Nuclear Envelope Marker (ab133741)

Lanes 1- 2: Merged signal (red and green). Green - ab133741 observed at 66-70 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab133741 was shown to react with LMNB1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255404 (knockout cell lysate ab263825) was used. Wild-type HeLa and LMNB1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab133741 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 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 in 20000 dilution for 1 hour at room temperature before imaging.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Ramos (Human Burkitt's lymphoma cell line) cells labeling Lamin B1 with ab133741 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/200 dilution (green). Nuclear envelope staining on Ramos cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:
-ve control 1: ab133741 at 1/100 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/200 dilution.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human transitional cell carcinoma of the bladder tissue labeling Lamin B1 with purified ab133741 at 1/300. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

ab133741 staining Lamin B1 in wild-type HAP1 cells (top panel) and Lamin B1 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% 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 with ab133741 at 1μg/ml dilution and ab195889 at 1/250 dilution (shown in pseudo-color red) overnight at +4°C. The cells were then incubated with ab150081 (Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488)) at 1/1000 dilution for 1 hour. Nuclear DNA was labelled in blue with DAPI.

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

Lanes 1 - 4: Merged signal (red and green). Green - ab133741 observed at 70 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab133741 was shown to specifically react with Lamin B1 in wild type HAP1 cells. No band was observed when knockout samples were used. Wild-type and Lamin B1 knockout samples were subjected to SDS-PAGE. Ab133741 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

Lanes 1 - 3: Merged signal (red and green).

Green - Target observed at 70 kDa. Red - loading control, ab8245, observed at 37 kDa.

This western blot image is a comparison between ab133741 and a competitor's discontinued goat polyclonal antibody.

Recombinant Human Lamin B1 protein (ab114163)

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Immunohistochemical staining of paraffin embedded Mouse Cerebral cortex with purified ab133741 at a working dilution of 1/300. The secondary antibody used is a HRP polymer for rabbit IgG. Nuclear envelope staining on neuron cells of Cerebral cortex tissue is observed. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labelling Lamin B1 with unpurified ab133741 at 1/250.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Blocking and Diluting buffer and concentration: 5% NFDM/TBST

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon tissue labelling Lamin B1 with unpurified ab133741 at 1/250.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

ab133741 (purified) at 1/20 immunoprecipitating Lamin B1 in Jurkat cells (Lane 1).  For western blotting, ab133741 was used at 1/1000 dilution and an HRP-conjugated goat anti-rabbit IgG was used as the secondary antibody (1/1500).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.