All lanes : Anti-L1CAM antibody [EPR23338-106] (ab272733) at 1/1000 dilution

Lane 1 : Mouse brain tissue lysate
Lane 2 : Rat brain tissue lysate
Lane 3 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
Lane 4 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000 dilution

Predicted band size: 140 kDa
Observed band size: 150,250 kDa why is the actual band size different from the predicted?

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

Negative control: NIH/3T3 (PMID: 22973895)

L1CAM is a glycoprotein. Full length 250-kDa L1CAM and cleaved 150-kDa are observed. The molecular weight observed is consistent with what have been described in literature (PMID: 20840789, PMID: 23205105).

Exposure time: 114 seconds.

 

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse cerebellum tissue labeling L1CAM with ab272733 at 1/500 dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 (2 ug/ml) dilution (Green). Positive staining on mouse cerebellum is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 (2 ug/ml) dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Immunofluorescent analysis of 100% methanol-fixed mouse primary neuron cell cells labelling L1CAM with ab272733 at 1/500 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing positive staining in mouse primary neuron cell. Confocal scanning Z step was set as 0.3 μm followed by image processing with maximum Z projection. L1CAM is specifically localized to axons, but was absent from MAP2-positive dendrites (PMID: 27001749). Negative control: NIH/3T3 (PMID: 22973895).  ab11267 Anti-MAP2 mouse monoclonal antibody was used to counterstain dentrites at 1/500 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) at 1/1000 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000  dilution.

Flow cytometric analysis of NIH/3T3 (Mouse embryonic fibroblast)(Left) / B16-F10 (Mouse melanoma mixture of spindle-shaped and epithelial-like cells)(Right) cells labelling L1CAM with ab272733 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Negative control: NIH/3T3 (PMID: 22973895).

Gated on viable cells.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat cerebellum tissue labeling L1CAM with ab272733 at 1/500 dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 (2 ug/ml) dilution (Green). Positive staining on rat cerebellum is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 (2 ug/ml) dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat kidney tissue labeling L1CAM with ab272733 at 1/500 dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 (2 ug/ml) dilution (Green). Positive staining on rat kidney is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 (2 ug/ml) dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Flow cytometric analysis of Mouse primary neuron cells cells labelling L1CAM with ab272733 at 1/500 dilution (0.1ug) (Right) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Left).

Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Gated on viable cells.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse kidney tissue labeling L1CAM with ab272733 at 1/500 dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 (2 ug/ml) dilution (Green). Positive staining on mouse kidney is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488)at 1/1000 (2 ug/ml) dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Flow cytometric analysis of PC-12 (Rat adrenal gland pheochromocytoma) cells labelling L1CAM with ab272733 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Gated on viable cells.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat colon tissue labeling L1CAM with ab272733 at 1/500 dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (Green). Positive staining on rat colon is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488)at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse colon tissue labeling L1CAM with ab272733 at 1/500 dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (Green). Positive staining on mouse colon is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Immunofluorescent analysis of 100% methanol-fixed PC-12 cells labelling L1CAM with ab272733 at 1/500 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000  dilution (Green). Confocal image showing membranous and cytoplasmic staining in PC-12 cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200  dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000  dilution.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat liver tissue labeling L1CAM with ab272733 at 1/100 dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 (2 ug/ml) dilution (Green). Negative control: No staining on rat liver (PMID: 22888955) is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 (2 ug/ml) dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse liver tissue labeling L1CAM with ab272733 at 1/100 dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 (2 ug/ml) dilution (Green). Negative control: No staining on mouse liver (PMID: 22888955) is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 (2 ug/ml) dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).