All lanes : Anti-L1CAM antibody [EPR18750] (ab208155) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : L1CAM knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 140 kDa

Lanes 1 - 2: Merged signal (red and green). Green - ab208155 observed at 220 kDa. Red - loading control, ab130007 observed at 125 kDa.

 ab208155 was shown to react with L1CAM in wild-type HeLa. Loss of signal was observed when knockout cell line ab255401 (knockout cell lysate ab263786) was used. Wild-type and L1CAM knockout samples were subjected to SDS-PAGE. ab208155 and Anti-Vinculin antibody [VIN-54] (ab130007) were incubated overnight at 4^°C at 1 in 1000 dilution and 1 in 20000 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 in 20000 dilution for 1 hour at room temperature before imaging.

 

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling L1CAM with ab208155 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasm staining on the mouse cerebrum is observed.
L1CAM specific staining most abundant on nervous system, distal kidney tubules, and tumor cells. [PMID: 16867862, PMID: 20044598].

Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

All lanes : Anti-L1CAM antibody [EPR18750] (ab208155) at 1/2000 dilution

Lane 1 : Human fetal brain lysate
Lane 2 : Human cerebellum lysate
Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 140 kDa
Observed band size: 140,200-220 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1 and 2: 30seconds; Lane 3: 3 minutes.

The product binds to the full length L1CAM and the 140KD fragment. Plasmin cleaves L1CAM at the FN3 repeat to produce 140 kDa and 85 kDa fragments (PMID: 7542658;PMID: 20840789). The 140 kDa fragment is where the immunogen is located.

All lanes : Anti-L1CAM antibody [EPR18750] (ab208155) at 1/2000 dilution

Lane 1 : Rat brain lysate
Lane 2 : Rat cerebellum lysate
Lane 3 : Rat hippocampus 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: 140,200-220 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The product binds to the full length L1CAM and the 140KD fragment. Plasmin cleaves L1CAM at the FN3 repeat to produce 140 kDa and 85 kDa fragments (PMID: 7542658; PMID: 20840789). The 140 kDa fragment is where the immunogen is located.

All lanes : Anti-L1CAM antibody [EPR18750] (ab208155) at 1/1000 dilution

Lane 1 : Mouse cerebellum lysate
Lane 2 : Mouse brain lysate
Lane 3 : A-375 (Human malignant melanoma cell line) whole cell lysate

Lysates/proteins at 10 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: 140,200-220 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The product binds to the full length L1CAM and the 140KD fragment. Plasmin cleaves L1CAM at the FN3 repeat to produce 140 kDa and 85 kDa fragments (PMID: 7542658;PMID: 20840789). The 140 kDa fragment is where the immunogen is located.

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling L1CAM with ab208155 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Membrane staining on a part of Human kidney tubules is observed.
L1CAM specific staining most abundant on nervous system, distal kidney tubules, and tumor cells. [PMID: 16867862, PMID: 20044598].

Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Human stomach cancer tissue labeling L1CAM with ab208155 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Membrane staining on the tumor cells of Human stomach cancer is observed.
L1CAM specific staining most abundant on nervous system, distal kidney tubules, and tumor cells. [PMID: 16867862, PMID: 20044598].

Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

 

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling L1CAM with ab208155 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Negative staining on the Human liver.

L1CAM specific staining most abundant on nervous system, distal kidney tubules, and tumor cells. [PMID: 16867862, PMID: 20044598].

Counter stained with Hematoxylin.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling L1CAM with ab208155 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Mainly membrane staining on the nerve tract of mouse colon is observed.
L1CAM specific staining most abundant on nervous system, distal kidney tubules, and tumor cells. [PMID: 16867862, PMID: 20044598].

Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling L1CAM with ab208155 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Negative staining on the mouse testis.
L1CAM specific staining most abundant on nervous system, distal kidney tubules, and tumor cells. [PMID: 16867862, PMID: 20044598].

Counter stained with Hematoxylin.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Rat cerebellum tissue labeling L1CAM with ab208155 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasm staining on the molecular layer of the rat cerebellar cortex is observed.
L1CAM specific staining most abundant on nervous system, distal kidney tubules, and tumor cells. [PMID: 16867862, PMID: 20044598].

Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Rat colon tissue labeling L1CAM with ab208155 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasm staining on the nerve tract of the rat colon is observed.
L1CAM specific staining most abundant on nervous system, distal kidney tubules, and tumor cells. [PMID: 16867862, PMID: 20044598].

Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Rat skeletal muscle tissue labeling L1CAM with ab208155 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Negative staining on the rat skeletal muscle.
L1CAM specific staining most abundant on nervous system, distal kidney tubules, and tumor cells. [PMID: 16867862, PMID: 20044598].

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

L1CAM was immunoprecipitated from 1mg of Human cerebellum lysate with ab208155 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab208155 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.
Lane 1: Human cerebellum lysate 10µg (Input).
Lane 2: ab208155 IP in Human cerebellum lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab208155 in Human cerebellum lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 3 minutes.

L1CAM was immunoprecipitated from 1mg of Rat brain whole cell lysate with ab208155 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab208155 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.
Lane 1: Rat brain whole cell lysate 10µg (Input).
Lane 2: ab208155 IP in Rat brain whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab208155 in Rat brain whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 3 minutes.