All lanes : Anti-CRMP1 antibody [EP14521] (ab199722) at 1/5000 dilution

Lane 1 : Human fetal brain lysate
Lane 2 : SH-SY5Y (Human neuroblastoma from bone marrow cells) cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 62 kDa
Observed band size: 64,80 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Based on the sequence analysis, ab199722 recognizes two isoforms with the predicted MWs of 62 kDa and 74 kDa, respectively.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) and Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling CRMP1 with ab199722 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Cytoplasm and nuclear staining on U-87 MG cell line is observed. Negative expression in Jurkat cells. The nuclear counterstain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).

The negative controls are as follows:-
-ve control 1: ab199722 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

 

All lanes : Anti-CRMP1 antibody [EP14521] (ab199722) at 1/1000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : C6 (Rat glial tumor cells) cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 62 kDa
Observed band size: 64 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking buffer: 5% NFDM/TBST.

Dilution buffer: 5% NFDM /TBST or 1%BSA /TBST.

Based on the sequence analysis, CRMP1 does not have similar isoforms within Mouse & Rat, therefore only a single band at approximately 62kDa is seen in Mouse and Rat lysates within western blot.

Immunohistochemical analysis of paraffin-embedded Human cerebral cortex tissue labeling CRMP1 with ab199722 at 1/8000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm and nucleus staining on Human cerebral cortex is observed. 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 cerebral cortex tissue labeling CRMP1 with ab199722 at 1/8000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on mouse cerebral cortex is observed. 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 cerebral cortex tissue labeling CRMP1 with ab199722 at 1/8000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on rat cerebral cortex is observed. 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 CRMP1 with ab199722 at 1/8000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. No staining on Human liver tissue is observed. 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.

Flow cytometric analysis of 2% paraformaldehyde-fixed SH-SY5Y (Human neuroblastoma from bone marrow cells) cells labeling CRMP1 with ab199722 at 1/20 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was  used as the secondary antibody.

CRMP1 was immunoprecipitated from 1mg of Human fetal brain whole cell lysate with ab199722 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab199722 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500.

Lane 1: Human fetal brain whole cell lysate 10ug (Input). Lane 2: ab199722 IP in Human fetal brain whole cell lysate. Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab199722 in Human fetal brain whole cell lysate.

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

Exposure time: 3 seconds.