All lanes : Anti-L1CAM antibody [EPR23241-224] (ab270455) at 1/1000 dilution

Lane 1 : Wild-type HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : Human L1CAM knockout HeLa cell line (ab255401)
Lane 3 : A375 (human malignant melanoma epithelial cell), whole cell lysate
Lane 4 : A549 (human lung carcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 140 kDa
Observed band size: 250 kDa

why is the actual band size different from the predicted?


Exposure time: 3 minutes

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

ab270455 was shown to specifically react with L1CAM in wild-type HeLa cells. Loss of signal was observed when knockout sample from Human L1CAM knockout HeLa cell line (ab255401) was used. Wild-type and L1CAM knockout samples were subjected to SDS-PAGE. ab270455 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument using the ECL technique.

A549 is negative or very low expression cell line for L1CAM (PMID: 23511563).

Immunohistochemical analysis of paraffin-embedded Human cerebellum tissue labeling L1CAM with ab270455 at 1/100 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on the molecular layer of the human cerebellum (PMID: 12514225). The section was incubated with ab270455 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunocytochemistry/Immunofluorescence analysis of A375 (human malignant melanoma cell line) cells labelling L1CAM with ab270455 at 1/100. Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) (ab150077) was used as the secondary antibody. The cells were counterstained with an Alexa Fluor^® 594-conjugated mouse anti-alpha tubulin antibody (1/200) (ab195889). Nuclei counterstained with DAPI (blue).

Confocal image showing strong membranous and weak cytoplasmic staining in A375 cells.

Flow cytometric analysis of L1CAM KO HeLa (Human cervix adenocarcinoma epithelial cell, Left) / Parental HeLa (Right) cells labeling L1CAM with ab270455 at 1/50 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled 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.

Human L1CAM knockout HeLa cell line (ab255401).
Gated on viable cells.

All lanes : Anti-L1CAM antibody [EPR23241-224] (ab270455) at 1/1000 dilution

Lane 1 : Human brain tissue lysate
Lane 2 : A375 (human malignant melanoma epithelial cell), whole cell lysate
Lane 3 : A549 (human lung carcinoma epithelial cell), whole cell lysate
Lane 4 : MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 140 kDa
Observed band size: 250,80 kDa why is the actual band size different from the predicted?

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

L1CAM is a glycoprotein. Full length 250-kDa L1CAM and cleaved 80-kDa are observed. The molecular weight observed is consistent with what have been described in literature (PMID 29127326).

A549 is negative or very low expression cell line for L1CAM (PMID: 23511563).

Exposure time: 3 minutes.

Immunocytochemistry/Immunofluorescence analysis of wild-type and L1CAM knockout HeLa (human epithelial cell line from cervix adenocarcinoma) cells labelling L1CAM with ab270455 at 1/500. Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) (ab150077) was used as the secondary antibody. The cells were counterstained with an Alexa Fluor^® 594-conjugated mouse anti-alpha tubulin antibody (1/200) (ab195889). Nuclei counterstained with DAPI (blue).

Confocal image showing strong membranous and weak cytoplasmic staining in wild-type HeLa cells, while no staining in L1CAM knockout HeLa cells (ab255401).

Flow cytometric analysis of A549 (Human lung carcinoma epithelial cell, Left) / A375 (Human malignant melanoma epithelial cell, Right) cells labelling L1CAM with ab270455 at 1/500 dilution (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.

A549 is negative or very low expression cell line for L1CAM (PMID: 23511563).

Gated on viable cells.

L1CAM was immunoprecipitated from 0.35 mg A375 (human malignant melanoma epithelial cell), whole cell lysate using ab270455 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab270455 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: A375 (human malignant melanoma epithelial cell), whole cell lysate 10ug

Lane 2: ab270455 IP in A375 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab270455 in A375 whole cell lysate

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

Exposure time: 5.5 seconds.

 

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling L1CAM with ab270455 at 1/100 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on the nerve in human spleen (PMID: 26743472). The section was incubated with ab270455 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling L1CAM with ab270455 at 1/100 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Membranous staining on distal renal tubules in human kidney. The section was incubated with ab270455 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.