All lanes : Anti-Lamin A + Lamin C antibody [WL4G10] (ab232730) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : Lamin A + C knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 74 kDa

Lanes 1-2: Merged signal (red and green). Green - ab232730 observed at 70,75 kDa. Red - loading control ab181602 observed at 37 kDa.

 ab232730 Anti-Lamin A + C antibody [WL4G10] was shown to specifically react with Lamin A + C in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab261787 (knockout cell lysate ab256979) was used. Wild-type and Lamin A + C knockout samples were subjected to SDS-PAGE. ab232730 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

 

 

ab232730 staining Lamin A+C (colored green) in wild-type HAP1 cells (top panel) and LMNA knockout HAP1 cells (bottom panel).

The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 mins 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 ab232730 at 1 μg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control, at 1/1000 dilution overnight at 4ºC. Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (shown in green) and ab150084, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolor red). Nuclear DNA was labeled with DAPI (shown in blue).

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

All lanes :

Lane 1 : HAP1 whole cell lysate
Lane 2 : HAP1 LMNA KO whole cell lysate
Lane 3 : HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 74 kDa
Observed band size: 70,75 kDa why is the actual band size different from the predicted?

ab232730 was shown to specifically react with LMNA (Lamin A + C) in wild type HAP1 cells. No band was observed when LMNA (Lamin A + C) knockout samples were used. Wild-type and LMNA (Lamin A + C) knockout samples were subjected to SDS-PAGE. ab232730 and ab181602 (Rabbit anti GAPDH) were incubated overnight at 4°C at a 1mg/ml concentration and 1/10000 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.

IHC image of Lamin A + C staining in a section of formalin-fixed paraffin-embedded normal human skin* performed on a Leica BOND^TM system using the standard protocol F.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab232730, 1 µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with hematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

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

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

ab232730 staining Lamin A+C in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 mins 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 ab232730 at 1 μg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control, at 1/1000 dilution. Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (shown in green) and ab150084, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolor red). Nuclear DNA was labeled with DAPI (shown in blue).