All lanes : Anti-Lamin A + Lamin C antibody [EPR4100] - Nuclear Envelope Marker (ab108595) at 1/10000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : Lamin A/C knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 63,74 kDa
Additional bands at: 64 kDa (possible cleavage fragment), 76 kDa (possible cleavage fragment)

ab108595 was shown to specifically react with Lamin A + C (LMNA) in wild type HAP1 cells. No band was observed when Lamin A + C (LMNA) knockout samples were used. Wild-type and Lamin A + C (LMNA) knockout samples were subjected to SDS-PAGE. The membrane was blocked for an hour using 5% milk before ab108595 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at a 1/10000 and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

Anti-Lamin A + Lamin C antibody [EPR4100] - Nuclear Envelope Marker (ab108595) at 1/100000 dilution (purified) + HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate at 10 µg

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

Predicted band size: 63,74 kDa
Observed band size: 63,74 kDa why is the actual band size different from the predicted?

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

Histological analyses of hiPSCs (Human induced pluripotent stem cell) transplanted into the subretinal space of nude rats.

Eye balls were excised from a nude rat 7 weeks after subretinal transplantation with 1×10⁴ hiPSCs. Transplanted tissues were fixed with 4% paraformaldehyde. Paraffin embedded tissue sections were stained with haematoxylin/eosin. Then, the paraffin sections were deparaffinized with xylene and sequential 100%, 95%, 80%, 70% ethanol treatments for 5 min each. The sections were treated with 10 mM citric acid (pH 6) at 95°C for 50 min followed by permeation with 0.4% Triton-X in PBS at room temperature for 30 min.

The deparaffinized sections were stained with ab108595 (Panel M), Hoechst 33258 (Panel N).

All lanes : Anti-Lamin A + Lamin C antibody [EPR4100] - Nuclear Envelope Marker (ab108595) at 1/100000 dilution (purified)

Lane 1 : HepG2 (Human liver hepatocellular carcinoma cell line) cell lysate
Lane 2 : HaCaT (Human keratinocyte cell line) cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 63,74 kDa
Observed band size: 63,74 kDa why is the actual band size different from the predicted?

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

All lanes : Anti-Lamin A + Lamin C antibody [EPR4100] - Nuclear Envelope Marker (ab108595) at 1/10000 dilution (unpurified)

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysates
Lane 2 : SH-SY5Y (Human neuroblastoma cell line from bone marrow) cell lysates

Lysates/proteins at 10 µg per lane.

Predicted band size: 63,74 kDa

Immunocytochemistry/Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Lamin A + C (green) with purified ab108595 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

Control: Primary antibody (1/500) and secondary antibody ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Immunocytochemsitry/Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Lamin A + C with unpurified ab108595 at 1/250 dilution.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervix carcinoma tissue labeling Lamin A + C with purified ab108595 at 1/250. 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. Counterstained with hematoxylin.

Negative control using PBS instead of primary antibody (inset).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human brain tissue labeling Lamin A + C with ab108595 at 1/250 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labeling Lamin A + C with ab108595 at 1/250 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue labeling Lamin A + C with ab108595.

Green - Lamin A + C.

Red - PI.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue labeling Lamin A + C with ab108595.

Green - Lamin A + C.

Red - PI.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human urothelial carcinoma tissue labeling Lamin A + C with ab108595.

Green - Lamin A + C.

Red - PI.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colonic carcinoma tissue labeling Lamin A + C with ab108595.

Green - Lamin A + C.

Red - PI.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

ab108595 (purified) at 1/30 immunoprecipitating Lamin A + C in HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate. For western blotting, a peroxidase-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/1000).

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

Flow Cytometry analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Lamin A + C with purified ab108595 at 1/110 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody.

Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabeled control, cells without incubation with primary and secondary antibodies.

Overlay histogram showing HeLa (Human epithelial cell line from cervix adenocarcinoma) cells stained with unpurified ab108595 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab108595, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabeled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.