All lanes : Anti-Lamin B1 antibody [EPR22165-121] (ab229025) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : LMNB1 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 66 kDa
Observed band size: 66-70 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab229025 observed at 66-70 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab229025 was shown to react with Lamin B1 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. ab229025 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.

Multiplex immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue.

Panel A:  Merged staining of Collagen VI (ab182744; green), anti-CD68 (ab213363; red) and anti-Lamin B1 (ab229025; magenta).

Panel B: Anti-Collagen VI (green) stained on extracellular matrix.

Panel C: Anti-CD68 (red) stained on Kupffer cells.

Panel D: Anti-Lamin B1 (magenta) stained on nuclear envelope.

Key protocol steps:  The section was incubated in three rounds of staining with ab182744 (1/1000 dilution), ab213363 (1/1000 dilution) and ab229025 (1/4000 dilution) for 30 mins at room temperature. Each round was followed by tyramide signal amplification with the appropriate fluorophore. Heat mediated antigen retrieval was used (Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins after every round of antibody/fluorophore staining.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

DAPI was used as a nuclear counter stain. A ready-to-use anti-Rabbit and Mouse Polymer HRP was used as a secondary.

All lanes : Anti-Lamin B1 antibody [EPR22165-121] (ab229025) at 1/5000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : Lamin B1 knockout HAP1 whole cell lysate
Lane 3 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate
Lane 4 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 66 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?


Exposure time: 8 seconds

ab229025 was shown to specifically react with Lamin B1 in wild-type HAP1 cells as signal was lost in Lamin B1 knockout cells. Wild-type and Lamin B1 knockout samples were subjected to SDS-PAGE. ab229025 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD^® ChemiDoc™ MP instrument using the ECL technique.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling Lamin B1 (Green) with ab229025 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution. Confocal image showing nuclear membranous staining in HeLa cells. Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) was used as a counterstain at 1/200 dilution. The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Alexa Fluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution.

 

Immunohistochemical analysis of paraffin-embedded mouse liver tissue stained for Lamin B1 using ab229025 at 1/2000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear envelope staining on mouse liver (PMID: 19925772) is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryonic fibroblast cell line) cells labeling Lamin B1 (Green) with ab229025 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution. Confocal image showing nuclear membranous staining in NIH/3T3 cells. Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) was used as a counterstain at 1/200 dilution. The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Alexa Fluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution.

 

All lanes : Anti-Lamin B1 antibody [EPR22165-121] (ab229025) at 1/1000 dilution

Lane 1 : Human lymph node tissue lysate
Lane 2 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 3 : HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 4 : Caco-2 (Human colorectal adenocarcinoma cell line) whole cell lysate
Lane 5 : Mouse brain tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lane 1 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/100000 dilution
Lanes 2-5 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 66 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?

Blocking/diluting buffer and concentration: 5% NFDM/TBST.

Exposure time:

Lane 1:3 seconds;

Lanes 2,3,4 and 5:10 seconds.

All lanes : Anti-Lamin B1 antibody [EPR22165-121] (ab229025) at 1/1000 dilution

Lane 1 : Mouse heart tissue lysate
Lane 2 : Rat brain tissue lysate
Lane 3 : Rat heart tissue lysate
Lane 4 : Rat spleen tissue lysate
Lane 5 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 6 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 7 : NIH/3T3 (Mouse embryo fibroblast cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 66 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?

Blocking/diluting buffer and concentration: 5% NFDM/TBST.

Exposure time:

Lane 1:10 seconds; Lanes 2 and 3: 59 seconds; Lane 4:3 seconds; Lanes 5,6 and 7:6 seconds.

Immunohistochemical analysis of paraffin-embedded rat cerebrum tissue stained for Lamin B1 using ab229025 at 1/2000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear envelope staining on rat cerebrum (PMID: 26469707, PMID: 19925772) is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded human breast tissue stained for Lamin B1 using ab229025 at 1/2000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear envelope staining on human breast (PMID: 26469707) is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).

Lamin B1 was immunoprecipitated from 0.35 mg HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab229025 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab229025 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1: HeLa whole cell lysate 10 μg (input).
Lane 2: ab229025 IP in HeLa whole cell lysate (+).
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab229025 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 5 seconds.

A very weak band around 66 kDa detected in lane 3 is due to spill over from lane2 (+).

Lamin B1 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate with ab229025 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab229025 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1: NIH/3T3 whole cell lysate 10 μg (input).
Lane 2: ab229025 IP in NIH/3T3 whole cell lysate (+).
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab229025 in NIH/3T3 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 5 seconds.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized NIH/3T3 (mouse embryo fibroblast cell line) cell line labeling Lamin B1 with ab229025 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG isotype control (ab172730) (Black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/2000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cell line labeling Lamin B1 with ab229025 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG isotype control (ab172730) (Black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/2000 dilution.