Anti-Lamin B1 antibody [EPR22165-121] - Nuclear Envelope Marker

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Lamin B1 antibody [EPR22165-121] - Nuclear Envelope Marker (AB229025)

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.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Lamin B1 antibody [EPR22165-121] - Nuclear Envelope Marker (AB229025)

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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Lamin B1 antibody [EPR22165-121] - Nuclear Envelope Marker (AB229025)

Intracellular 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 IgGisotype 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lamin B1 antibody [EPR22165-121] - Nuclear Envelope Marker (AB229025)

Immunohistochemical analysis of paraffin-embedded human endometrial carcinoma stained for Lamin B1 using ab229025 at 1/2000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Nuclear envelope staining on human endometrial carcinoma is observed. Counterstained with hematoxylin. Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). The section was incubated with ab229025 at 4°C overnight

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Lamin B1 antibody [EPR22165-121] - Nuclear Envelope Marker (AB229025)

ab229025 staining LMNB1 in HeLa WT and HeLa LMNB1 KO 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 ab229025 at 0.2µg/ml (shown in green) and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control (shown in pseudocolour magenta). Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution. 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 single confocal section is shown.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Lamin B1 antibody [EPR22165-121] - Nuclear Envelope Marker (AB229025)

ab229025 staining LMNB1 in Hap1 WT and Hap1 LMNB1 KO 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 ab229025 at 0.2µg/ml (shown in green) and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control (shown in pseudocolour magenta). Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution. 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 single confocal section is shown.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lamin B1 antibody [EPR22165-121] - Nuclear Envelope Marker (AB229025)

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).

• IP

Supplier Data

Immunoprecipitation - Anti-Lamin B1 antibody [EPR22165-121] - Nuclear Envelope Marker (AB229025)

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 (+).

All lanes:

Immunoprecipitation - Anti-Lamin B1 antibody [EPR22165-121] - Nuclear Envelope Marker (ab229025)

Predicted band size: 66 kDa

Observed band size: 66 kDa

false

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lamin B1 antibody [EPR22165-121] - Nuclear Envelope Marker (AB229025)

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).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Lamin B1 antibody [EPR22165-121] - Nuclear Envelope Marker (AB229025)

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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Lamin B1 antibody [EPR22165-121] - Nuclear Envelope Marker (AB229025)

Intracellular 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 aRabbit monoclonal IgGisotype 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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lamin B1 antibody [EPR22165-121] - Nuclear Envelope Marker (AB229025)

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).

• IP

Supplier Data

Immunoprecipitation - Anti-Lamin B1 antibody [EPR22165-121] - Nuclear Envelope Marker (AB229025)

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.

All lanes:

Immunoprecipitation - Anti-Lamin B1 antibody [EPR22165-121] - Nuclear Envelope Marker (ab229025)

Predicted band size: 66 kDa

Observed band size: 66 kDa

false

• WB

Supplier Data

Western blot - Anti-Lamin B1 antibody [EPR22165-121] - Nuclear Envelope Marker (AB229025)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-Lamin B1 antibody [EPR22165-121] (ab229025) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-Histone H3 antibody [RM1288] - Nuclear Marker (<a href='/en-us/products/primary-antibodies/histone-h3-antibody-rm1288-nuclear-marker-ab322707'>ab322707</a>) at 1/10000 dilution

Lane 1:

HEK-293 (human embryonic kidney epithelial cell) nuclear fraction at 10 µg

Lane 2:

HEK-293 cytoplasmic fraction at 10 µg

Lane 3:

NIH/3T3 (mouse embryonic fibroblast) nuclear fraction at 10 µg

Lane 4:

NIH/3T3 cytoplasmic fraction at 10 µg

Lane 5:

Rat brain tissue nuclear fraction at 10 µg

Lane 6:

Rat brain tissue cytoplasmic fraction at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 16 kDa,14 kDa,36 kDa,66 kDa

false

Exposure time: 8s

• WB

Lab

Western blot - Anti-Lamin B1 antibody [EPR22165-121] - Nuclear Envelope Marker (AB229025)

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.

All lanes:

Western blot - Anti-Lamin B1 antibody [EPR22165-121] - Nuclear Envelope Marker (ab229025) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

LMNB1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human LMNB1 (Lamin B1) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-lmnb1-lamin-b1-knockout-hela-cell-line-ab255404'>ab255404</a>)

Predicted band size: 66 kDa

Observed band size: 66-70 kDa

false

• WB

Supplier Data

Western blot - Anti-Lamin B1 antibody [EPR22165-121] - Nuclear Envelope Marker (AB229025)

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

Exposure time :

Lane 1 : 3 seconds;

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

All lanes:

Western blot - Anti-Lamin B1 antibody [EPR22165-121] - Nuclear Envelope Marker (ab229025) at 1/1000 dilution

Lane 1:

Human lymph node tissue lysate at 20 µg

Lane 2:

HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 3:

HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg

Lane 4:

Caco-2 (Human colorectal adenocarcinoma cell line) whole cell lysate at 20 µg

Lane 5:

Mouse brain tissue lysate at 20 µg

Secondary

Lane 1:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/100000 dilution

Lanes 2 - 5:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 66 kDa

Observed band size: 70 kDa

false

• WB

Lab

Western blot - Anti-Lamin B1 antibody [EPR22165-121] - Nuclear Envelope Marker (AB229025)

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.

All lanes:

Western blot - Anti-Lamin B1 antibody [EPR22165-121] - Nuclear Envelope Marker (ab229025) at 1/5000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

Lamin B1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

Predicted band size: 66 kDa

Observed band size: 70 kDa

false

Exposure time: 8s

• WB

Supplier Data

Western blot - Anti-Lamin B1 antibody [EPR22165-121] - Nuclear Envelope Marker (AB229025)

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.

All lanes:

Western blot - Anti-Lamin B1 antibody [EPR22165-121] - Nuclear Envelope Marker (ab229025) at 1/1000 dilution

Lane 1:

Mouse heart tissue lysate at 10 µg

Lane 2:

Rat brain tissue lysate at 10 µg

Lane 3:

Rat heart tissue lysate at 10 µg

Lane 4:

Rat spleen tissue lysate at 10 µg

Lane 5:

RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg

Lane 6:

PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Lane 7:

NIH/3T3 (Mouse embryo fibroblast cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 66 kDa

Observed band size: 70 kDa

false