Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Lamin A knockout HAP1 cell lysate (20 µg)
Lane 3: A431 cell lysate (20 µg)
Lane 4: NIH3T3 cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab26300 observed at 76 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab26300 was shown to recognize Lamin A in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when Lamin A knockout samples were examined. Wild-type and Lamin A knockout samples were subjected to SDS-PAGE. ab26300 1ug/ml and ab8245 (loading control to GAPDH) at a dilution of 1/1000 were incubated overnight at 4°C. 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 1/10,000 dilution for 1 hour at room temperature before imaging.

ab26300 stained in Hela cells. Cells were fixed with 100% methanol (5 min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab26300 at 1µg/ml and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150120 (pseudo-colored red) and ab150081 (colored green) used at 1 ug/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature.

Immunocytochemistry/ Immunofluorescence analysis of hESCs labeling Lamin A with ab26300 at 1/500 dilution. Samples were fixed with 3.7% paraformaldehyde for 1 hour, and stained for nuclear DNA (DAPI), filamentous actin, tumor recognition antigen 1–81, and nuclear envelope protein Lamin A. For staining, cells were permeabilized with 0.1% Triton X-100 for 10 min. Goat serum, 10%, in phosphate-buffered saline was used to block nonspecific binding for 20 min.

 ab26300 (1/2000) staining Lamin A in assynchronous HeLa Cells, by Immunocytochemistry/ Immunofluorescence. Secondary antibody: goat anti-rabbit conjugated to Cy3 ^® (1/200). Cells counterstained with DAPI in order to highlight the nucleus.

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Anti-Lamin A antibody (ab26300) at 1/1000 dilution + HeLa whole cell extract at 100 µg

Secondary
Goat anti-Rabbit IgG (H+L) HRP Conjugate at 1/10000 dilution

Developed using the ECL technique.

Predicted band size: 74 kDa
Observed band size: 76 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

Blocking: 5% milk for 30 minutes at 22°C 

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Immunocytochemistry/ Immunofluorescence analysis of human vascular smooth muscle cell labeling Lamin A with ab26300 at 1/200 dilution. Cells were fixed in formaldehyde and permeabilized with np40. Cells were blocked with 3% BSA for 1 hour at 21°C. A polyclonal donkey anti-rabbit Alexa Fluor^® 568 conjugated secondary antibody was used at 1/500 dilution.

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All lanes : Anti-Lamin A antibody (ab26300) at 1/1000 dilution

Lane 1 : Mouse NIH-3T3 cells - cytosolic fraction
Lane 2 : Mouse NIH-3T3 cells - nuclear fraction

Lysates/proteins at 25 µg per lane.

Secondary
All lanes : HRP conjugated Goat anti-rabbit at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 74 kDa
Observed band size: 76 kDa why is the actual band size different from the predicted?


Exposure time: 10 seconds

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Immunocytochemistry/ Immunofluorescence analysis of Human Lung Fibroblasts labeling Lamin A with ab26300 at 1/500 dilution. Samples were fixed with 3.7% paraformaldehyde for 1 hour, and stained for nuclear DNA (DAPI), filamentous actin, tumor recognition antigen 1–81, and nuclear envelope protein Lamin A. For staining, cells were permeabilized with 0.1% Triton X-100 for 10 min. Goat serum, 10%, in phosphate-buffered saline was used to block nonspecific binding for 20 min.

Anti-Lamin A antibody (ab26300) at 1 µg/ml + A-431 whole cell lysate (ab7909) at 20 µg

Secondary
IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/15000 dilution

Performed under reducing conditions.

Predicted band size: 74 kDa
Observed band size: 76 kDa why is the actual band size different from the predicted?
Additional bands at: 68 kDa (possible degradation product)

All lanes : Anti-Lamin A antibody (ab26300) at 1 µg/ml

Lane 1 : Recombinant Human Lamin A protein (ab83472) at 0.1 µg
Lane 2 : Recombinant Human Lamin A protein (ab83472) at 0.01 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 74 kDa


Exposure time: 10 seconds

ab26300 at 1/1000 staining human HeLa cells by ICC/IF. The cells were paraformaldehyde fixed, permeabilized with Triton X100 and blocked with BSA before incubation with the antibody. A Cy3 ® conjugated donkey anti-rabbit IgG was used as the secondary.

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All lanes : Anti-Lamin A antibody (ab26300) at 1 µg/ml

Lane 1 : NIH/3T3 whole cell lysate (ab7179)
Lane 2 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 74 kDa
Observed band size: 74 kDa
Additional bands at: 100 kDa, 45 kDa, 70 kDa (possible degradation product). We are unsure as to the identity of these extra bands.