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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Lamin B1 antibody [EPR8985(B)] - Nuclear Envelope Marker (AB133741)

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).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lamin B1 antibody [EPR8985(B)] - Nuclear Envelope Marker (AB133741)

Immunohistochemical analysis of formalin fixed paraffin embedded human colon labelling Lamin B1 with ab133741 at a concentration of 0.1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab133741 anti-Lamin B1 antibody [EPR8985(B)] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lamin B1 antibody [EPR8985(B)] - Nuclear Envelope Marker (AB133741)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lamin B1 antibody [EPR8985(B)] - Nuclear Envelope Marker (AB133741)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lamin B1 antibody [EPR8985(B)] - Nuclear Envelope Marker (AB133741)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Lamin B1 antibody [EPR8985(B)] - Nuclear Envelope Marker (AB133741)

ab133741 staining LMNB1 in HeLa WT and HeLa LMNB1 KO cells. The cells were fixed with 100% methanol (5 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 ab133741 at 1µg/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).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a single confocal section is shown.

• IP

Lab

Immunoprecipitation - Anti-Lamin B1 antibody [EPR8985(B)] - Nuclear Envelope Marker (AB133741)

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.

All lanes:

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

Predicted band size: 66 kDa

Observed band size: 70 kDa

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• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lamin B1 antibody [EPR8985(B)] - Nuclear Envelope Marker (AB133741)

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.

• WB

Lab

Western blot - 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.

All lanes:

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

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• WB

Lab

Western blot - Anti-Lamin B1 antibody [EPR8985(B)] - Nuclear Envelope Marker (AB133741)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Western blot - Anti-Lamin B1 antibody [EPR8985(B)] - Nuclear Envelope Marker (ab133741) at 1/10000 dilution

All lanes:

Jurkat (Human T cell leukemia cells from peripheral blood) cell lysate at 10 µg

Secondary

All lanes:

Peroxidase conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 66 kDa

Observed band size: 70 kDa

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• WB

Lab

Western blot - Anti-Lamin B1 antibody [EPR8985(B)] - Nuclear Envelope Marker (AB133741)

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 and 3:

Western blot - Anti-Lamin B1 antibody [EPR8985(B)] - Nuclear Envelope Marker (ab133741) at 1/1000 dilution

Lanes 2 and 4:

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

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lanes 2 and 4:

Empty

Lane 3:

LMNB1 knockout HAP1 whole cell lysate at 20 µg

Predicted band size: 66 kDa

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• WB

Lab

Western blot - Anti-Lamin B1 antibody [EPR8985(B)] - Nuclear Envelope Marker (AB133741)

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.

All lanes:

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

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

Empty

Lane 3:

Lamin B1 knockout HAP1 cell lysate at 20 µg

Predicted band size: 66 kDa

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• WB

Lab

Western blot - Anti-Lamin B1 antibody [EPR8985(B)] - Nuclear Envelope Marker (AB133741)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Lamin B1 antibody [EPR8985(B)] - Nuclear Envelope Marker (ab133741) at 1/50000 dilution

Lane 1:

Jurkat (Human T cell leukemia cells from peripheral blood) cell lysate at 20 µg

Lane 2:

Molt-4 (Human lymphoblastic leukemia cell line) cell lysate at 20 µg

Lane 3:

Y79 (Human retinoblastoma cell line) cell lysate at 20 µg

Lane 4:

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

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 66 kDa

Observed band size: 70 kDa

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• WB

Supplier Data

Western blot - Anti-Lamin B1 antibody [EPR8985(B)] - Nuclear Envelope Marker (AB133741)

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

All lanes:

Western blot - Anti-Lamin B1 antibody [EPR8985(B)] - Nuclear Envelope Marker (ab133741) at 1/10000 dilution

Lane 1:

Mouse brain lysates at 10 µg

Lane 2:

Mouse heart lysates at 10 µg

Lane 3:

Mouse kidney lysates at 10 µg

Lane 4:

Mouse spleen lysates at 10 µg

Lane 5:

Rat brain lysates at 10 µg

Lane 6:

Rat heart lysates at 10 µg

Lane 7:

Rat spleen lysates at 10 µg

Lane 8:

C6 (Rat glial tumor cells) whole cell lysates at 10 µg

Lane 9:

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

Lane 10:

NIH/3T3 (Mouse embyro fibroblast cells) at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 66 kDa

Observed band size: 70 kDa

false

• WB

Supplier Data

Western blot - Anti-Lamin B1 antibody [EPR8985(B)] - Nuclear Envelope Marker (AB133741)

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

Negative control : WEHI-231 (PMID : 2842066).

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

Alternatively spliced forms of the EVI-1 gene encode at least three distinct proteins : EVI-1 (145 kDa), MDS1/EVI-1 (200 kDa) and EVI-1Δ324 (88 kDa) PMID : (PMID : 24944602).

The identity of the higher MW band at approximately 300 kDa is unknown.

In Western blot, Anti-Lamin B1 antibody [EPR8985 (B)] (ab133741) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-EVI1 antibody [EPR26816-18] (<a href='/en-us/products/primary-antibodies/evi1-antibody-epr26816-18-ab315973'>ab315973</a>) at 1/1000 dilution

Lane 1:

MEF (mouse embryo fibroblast) whole cell lysate at 60 µg

Lane 2:

WEHI-231 (mouse B cell lymphoma B lymphocyte) whole cell lysate at 60 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 1/10000 dilution

Observed band size: 75 kDa,200 kDa,70 kDa

false

Exposure time: 59s

• WB

Lab

Western blot - Anti-Lamin B1 antibody [EPR8985(B)] - Nuclear Envelope Marker (AB133741)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Lamin B1 antibody [EPR8985(B)] - Nuclear Envelope Marker (ab133741) at 1/50000 dilution

Lane 1:

C6 (Rat glial tumor cells) cell lysate at 20 µg

Lane 2:

PC12 (Rat adrenal gland pheochromocytoma) cell lysate at 20 µg

Lane 3:

NIH/3T3 (Mouse embyro fibroblast cells) cell lysate at 20 µg

Lane 4:

RAW264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) cell lysate at 20 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 66 kDa

Observed band size: 70 kDa

false

• WB

Lab

Western blot - Anti-Lamin B1 antibody [EPR8985(B)] - Nuclear Envelope Marker (AB133741)

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

All lanes:

Western blot - Anti-Lamin B1 antibody [EPR8985(B)] - Nuclear Envelope Marker (ab133741) at 1/1000 dilution

All lanes:

GST-tagged Recombinant Human Lamin B1 protein (aa 1 to 586) at 0.015 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 1/20000 dilution

Predicted band size: 66 kDa

Observed band size: 100 kDa

false

Exposure time: 1s

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-Lamin B1 antibody [EPR8985(B)] - Nuclear Envelope Marker (AB133741)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.