All lanes : Anti-Lamin A + Lamin B1 + Lamin C antibody [EPR4068] (ab108922) at 1/10000 dilution (purified)

Lane 1 : mouse heart lysate
Lane 2 : rat heart lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit IgG (H+L) at 1/100000 dilution

Predicted band size: 74 kDa
Additional bands at: 65 kDa (possible isoform), 70 kDa (possible isoform), 74 kDa (possible isoform)

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

Immunofluorescence staining of HeLa cells with purified ab108922 at a working dilution of 1/250, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab108922 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

Immunohistochemical staining of paraffin embedded rat liver with purified ab108922 at a working dilution of 1/200. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

All lanes : Anti-Lamin A + Lamin B1 + Lamin C antibody [EPR4068] (ab108922) at 1/1000 dilution

Lane 1 : Recombinant Human Lamin A protein (ab83472)
Lane 2 : GST tagged recombinant Human Lamin B1 protein (1 to 586) (91 KDa)

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 74 kDa
Observed band size: 75, 91 kDa

why is the actual band size different from the predicted?


Exposure time: 180 seconds

Blocking/Diluting buffer and concentration 5% NFDM/TBST

Lane 1 : Anti-Lamin A + Lamin B1 + Lamin C antibody [EPR4068] (ab108922) at 1/1000 dilution
Lane 2 : Anti-Myc tag antibody at 1/10000 dilution
Lane 3 : Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606) at 1/5000 dilution

All lanes : Myc and DDK tagged recombinant Human Lamin B2 protein

Lysates/proteins at 0.01 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 74 kDa
Observed band size: 68 kDa why is the actual band size different from the predicted?


Exposure time: 7 seconds

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

ab108922 does not cross react with Lamin B2.

Flow Cytometry analysis of HeLa cells labelling Lamin A + B1 + C with purified ab108922 at a dilution of 1/20 (red). Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. An Alexa Flour^® 488-conjugated goat anti-rabbit IgG (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

ab108922 (purified) at 1/20 immunoprecipitating Lamin A + B1 + C in 10 μg HepG2 (Lanes 1 and 2, observed at 70, 74, and 65 kDa - ab108922 recognises three isoforms). Lane 3 - PBS. For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

Blocking buffer and concentration: 5% NFDM/TBST.

Dilution buffer and concentration: 5% NFDM/TBST.

Anti-Lamin A + Lamin B1 + Lamin C antibody [EPR4068] (ab108922) at 1/10000 dilution (purified) + HeLa cell lysate at 10 µg

Secondary
HRP goat anti-rabbit IgG (H+L) at 1/100000 dilution

Predicted band size: 74 kDa
Additional bands at: 65 kDa (possible isoform), 70 kDa (possible isoform), 74 kDa (possible isoform)

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

Immunohistochemical staining of paraffin embedded human ovarian carcinoma with purified ab108922 at a working dilution of 1/200. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunohistochemical staining of paraffin embedded human liver carcinoma with purified ab108922 at a working dilution of 1/200. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunohistochemical staining of paraffin embedded mouse liver with purified ab108922 at a working dilution of 1/200. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunohistochemical analysis of paraffin-embedded human colonic tissue using unpurified ab108922 at a diltion of 1/100

All lanes : Anti-Lamin A + Lamin B1 + Lamin C antibody [EPR4068] (ab108922) at 1/1000 dilution (unpurified)

Lane 1 : HeLa cell lysate
Lane 2 : HepG2 cell lysate
Lane 3 : HACAT cell lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 74 kDa

Unpurified ab108922 showing positive staining in Normal human breast tissue.

Unpurified ab108922 showing positive staining in Normal human uterus tissue.

Unpurified ab108922 showing positive staining in Normal human kidney tissue.