Anti-Lamin A + Lamin C antibody [EPR4100] - Nuclear Envelope Marker - Mouse IgG2a (Chimeric)

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lamin A + Lamin C antibody [EPR4100] - Nuclear Envelope Marker - Mouse IgG2a (Chimeric) (AB322991)

Immunofluorescence staining of LMNA in sections of formalin-fixed paraffin-embedded human colon*.

Performed on a Leica BOND. The section was pre-treated using heat mediated antigen retrieval with sodium citrate (pH6.0) for 20 minutes. The section was then incubated at room temperature for 1 hour with ab322991 at 1/500 dilution and then incubated for 1 hour with ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue). The section was then mounted using Dako Fluorescence Mounting Medium®.

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

For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antigen retrieval conditions, antibody concentrations and incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Lamin A + Lamin C antibody [EPR4100] - Nuclear Envelope Marker - Mouse IgG2a (Chimeric) (AB322991)

ab322991 staining LMNA in Hap1 (positive) and Hap1-LMNA KO (negative) 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 ab322991 at 1µg/ml (shown in green) and ab202272, Alexa Fluor® 594 Rabbit monoclonal [EP1332Y] to alpha Tubulin - Microtubule Marker (shown in pseudocolour magenta). Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed 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 maximum intensity projection of confocal sections is shown.

• WB

Lab

Western blot - Anti-Lamin A + Lamin C antibody [EPR4100] - Nuclear Envelope Marker - Mouse IgG2a (Chimeric) (AB322991)

False colour image of Western blot : Anti-Lamin A + Lamin C antibody [EPR4100] - Nuclear Envelope Marker - Mouse IgG2a (Chimeric) (ab322991) staining at 1/10000 dilution, shown in black; Rabbit anti-GAPDH loading control staining, shown in magenta. In Western blot, ab322991 was shown to bind specifically to LMNA. A band was observed at 74 and 69 kDa in Wild-type HeLa and Hap1 cell lysates, with no signal observed at this size in LMNA knockout Hap1 cell lysates. Samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat Anti-Mouse IgG Fc (HRP) preadsorbed (ab98717) at 1 : 10000 and Goat anti-Rabbit IgG H&L 680RD at 1 : 20000.

All lanes:

Western blot - Anti-Lamin A + Lamin C antibody [EPR4100] - Nuclear Envelope Marker - Mouse IgG2a (Chimeric) (ab322991) at 1/10000 dilution

Lane 1:

Wild-type HAP1 cell lysate

Lane 2:

Wild-type HeLa cell lysate

Lane 3:

LMNA Knockout Hap1 cell lysate

Secondary

Lanes 1 - 3:

Western blot - Goat Anti-Mouse IgG Fc (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-fc-hrp-preadsorbed-ab98717'>ab98717</a>) at 1/10000 dilution

Lanes 1 - 3:

Goat anti-Rabbit IgG H&L 680RD

Observed band size: 74 kDa,69 kDa

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