Anti-Lamin C antibody [EPR28295-16] - Nuclear Envelope Marker

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lamin C antibody [EPR28295-16] - Nuclear Envelope Marker (AB314500)

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Lamin C with ab314500 at 1/2000 (0.258 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human colon (PMID : 21621406). The section was incubated with ab314500 for 30mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lamin C antibody [EPR28295-16] - Nuclear Envelope Marker (AB314500)

Immunohistochemical analysis of paraffin-embedded human colorectal carcinoma tissue labeling Lamin C with ab314500 at 1/2000 (0.258 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human colorectal carcinoma (PMID : 21621406). The section was incubated with ab314500 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Lamin C antibody [EPR28295-16] - Nuclear Envelope Marker (AB314500)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized LMNA KO HeLa (LMNA knockout human cervical adenocarcinoma epithelial cell) cells labelling Lamin C with ab314500 at 1/250 (2.06 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1002 (2ug/ml) dilution (Green). Confocal image showing nuclear staining in parental HeLa cells, and no staining in LMNA knockout HeLa cells. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1002 (2ug/ml) dilution.

• IP

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Immunoprecipitation - Anti-Lamin C antibody [EPR28295-16] - Nuclear Envelope Marker (AB314500)

Lamin C was immunoprecipitated from 0.35 mg HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate with ab314500 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab314500 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate Lane 2 : ab314500 IP in HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab314500 in HepG2 whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-Lamin C antibody [EPR28295-16] - Nuclear Envelope Marker (ab314500) at 1/30 dilution

All lanes:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 6s

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Lamin C antibody [EPR28295-16] - Nuclear Envelope Marker (AB314500)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling Lamin C with ab314500 at 1/250 (2.06 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1003 (2ug/ml) dilution (Green). Confocal image showing nuclear staining in NIH/3T3 cells. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1003 (2ug/ml) dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lamin C antibody [EPR28295-16] - Nuclear Envelope Marker (AB314500)

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling Lamin C with ab314500 at 1/2000 (0.258 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on mouse cerebrum (PMID : 22308344). The section was incubated with ab314500 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lamin C antibody [EPR28295-16] - Nuclear Envelope Marker (AB314500)

Immunohistochemical analysis of paraffin-embedded rat liver tissue labeling Lamin C with ab314500 at 1/2000 (0.258 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on rat liver. The section was incubated with ab314500 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• WB

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Western blot - Anti-Lamin C antibody [EPR28295-16] - Nuclear Envelope Marker (AB314500)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Negative control : Daudi, HL-60, Raji (PMID : 15867203). In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution. Exposure time : Lanes 1-5 : 81 seconds； Lanes 6-8 : 15 seconds.

All lanes:

Western blot - Anti-Lamin C antibody [EPR28295-16] - Nuclear Envelope Marker (ab314500) at 1/1000 dilution

Lane 1:

A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

Raji (human burkitts lymphoma b lymphocyte) whole cell lysate at 20 µg

Lane 4:

Daudi (human burkitts lymphoma lymphoblast) whole cell lysate at 20 µg

Lane 5:

HL-60 (human acute promyelocytic leukemia promyeloblast) whole cell lysate at 20 µg

Lane 6:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 7:

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

Lane 8:

L-929 (mouse connective tissue fibroblast) whole cell lysate at 20 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: 65 kDa

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• WB

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Western blot - Anti-Lamin C antibody [EPR28295-16] - Nuclear Envelope Marker (AB314500)

Blocking and diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of TBS Lysates at 20 µg per lane. Western blot : Anti-Lamin C antibody (ab314500) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab314500 was shown to bind specifically to Lamin C. Target of interest was observed at 65 kDa in wild-type HeLa cell lysates (lane 1) with no signal observed at this size in LMNA knockout cell line (lane 2, knockout cell line ab261787 / knockout cell lysate ab256979). To generate this image, samples were first run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in a fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed in TBS-T, incubated with secondary antibodies Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution for 1 h at room temperature, washed again then imaged. Negative control : Jurkat (PMID : 15867203).

All lanes:

Western blot - Anti-Lamin C antibody [EPR28295-16] - Nuclear Envelope Marker (ab314500) at 1/1000 dilution

Lane 1:

Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

LMNA knockout HeLa whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

Jurkat (human t cell leukemia t lymphocyte from peripheral blood) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (800CW) and Goat Anti-Mouse IgG H&L (680RD) at 1/20000 dilution

Observed band size: 63 kDa

false

• WB

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Western blot - Anti-Lamin C antibody [EPR28295-16] - Nuclear Envelope Marker (AB314500)

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

All lanes:

Western blot - Anti-Lamin C antibody [EPR28295-16] - Nuclear Envelope Marker (ab314500) at 1/1000 dilution

Lane 1:

Human colon cancer tissue lysate at 20 µg

Lane 2:

Mouse brain tissue lysate at 20 µg

Lane 3:

Mouse heart tissue lysate at 20 µg

Lane 4:

Rat brain tissue lysate at 20 µg

Lane 5:

Rat heart tissue lysate at 20 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: 65 kDa

false

Exposure time: 125s

• Dot

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Dot Blot - Anti-Lamin C antibody [EPR28295-16] - Nuclear Envelope Marker (AB314500)

Dot blot analysis of Lamin C using ab314500 at 1/1000 (0.515 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100,000 dilution.

Lane 1 : Lamin C peptide
Lane 2 : Lamin A peptide

Exposure time : 180 seconds

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

This antibody does not cross-react with human Lamin A.