Immunohistochemical analysis of paraffin-embedded mouse stomach tissue labeling NCAM1 with ab220360 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP), Ready to use. Positive staining of ganglion (arrow) in mouse stomach (PMID: 1705171). Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rab it IgG H&L (HRP), Ready to use.

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

All lanes : Anti-NCAM1 antibody [EPR21827] (ab220360) at 1/1000 dilution

Lane 1 : Neuro-2a (mouse neuroblastoma cell line) whole cell lysate
Lane 2 : L-929 (mouse connective tissue fibroblast cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

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

Developed using the ECL technique.

Predicted band size: 95 kDa
Observed band size: 140 kDa why is the actual band size different from the predicted?


Exposure time: 70 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Negative control: L-929 (PMID: 9696812)

All lanes : Anti-NCAM1 antibody [EPR21827] (ab220360) at 1/5000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse cerebellum lysate
Lane 3 : Rat brain lysate
Lane 4 : Rat cerebellum lysate

Lysates/proteins at 20 µg per lane.

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

Developed using the ECL technique.

Predicted band size: 95 kDa
Observed band size: 120,140,180 kDa why is the actual band size different from the predicted?

Exposure time: Lanes 1-2: 26 seconds; Lane 3: 70 seconds; Lane 4: 3 minutes.

Blocking/Dilution buffer: 5% NFDM/TBST.

The 120,140 and 180kDa bands are different isoforms as reported in the literature (PMID:26288071).

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling NCAM1 with ab220360 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP), Ready to use. Positive staining on mouse cerebrum (PMID: 1705171). Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rab it IgG H&L (HRP), Ready to use.

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling NCAM1 with ab220360 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP), Ready to use. Positive staining of ganglia (arrows) in rat colon (PMID: 1705171). Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rab it IgG H&L (HRP), Ready to use.

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 100% methanol-fixed, 0.1% Triton X-100 permeabilized Neuro-2a (mouse neuroblastoma cell line) cells labeling NCAM1 with ab220360 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous staining in Neuro-2a cell line.

Negative control: L-929 PMID: 9696812).

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) at 1/1000 dilution.

Flow cytometric analysis of L-929 (mouse connective tissue fibroblast cell line) cell line (left panel) and Neuro-2a (mouse neuroblastoma cell line) cell line (right panel) labeling NCAM1 with ab220360 at 1/500 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody. 

Gated on viable cells. Negative control: L-929.

NCAM1 was immunoprecipitated from 0.35 mg of mouse brain lysate with ab220360 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab220360 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1: Mouse brain lysate 10 µg (Input). 

Lane 2: ab220360 IP in mouse brain lysate. 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab220360 in mouse brain lysate.

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

Exposure time: 1 second.

The 120,140 and 180kDa bands are different isoforms as reported in the literature (PMID: 26288071).

NCAM1 was immunoprecipitated from 0.35 mg of rat brain lysate with ab220360 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab220360 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1: Rat brain lysate 10 µg (Input). 

Lane 2: ab220360 IP in rat brain lysate. 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab220360 in rat brain lysate.

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

Exposure time: 1 second.

The 120,140 and 180kDa bands are different isoforms as reported in the literature (PMID: 26288071).