All lanes : Anti-DCC antibody [EPR23313-115] (ab273570) at 1/1000 dilution

Lane 1 : Human hippocampus tissue lysate
Lane 2 : SW480 (human colorectal adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : Mouse P5 hippocampus tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : VeriBlot for IP secondary antibody(HRP)(ab131366) at 1/1000 dilution

Predicted band size: 158 kDa
Observed band size: 175 kDa why is the actual band size different from the predicted?

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

DCC is a glycoprotein. The molecular weight observed is consistent with what have been described in the literature (PMID: 8044801).

Negative control: SW480 (PMID: 7926722).

Exposure time: 3 minutes.

Immunohistochemical analysis of paraffin-embedded Mouse E14.5 tissue labeling DCC with ab273570 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on mouse E14.5 cerebrum (PMID: 24739528). The section was incubated with ab273570 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.

All lanes : Anti-DCC antibody [EPR23313-115] (ab273570) at 1/1000 dilution

Lane 1 : Rat P7 hippocampus tissue lysate
Lane 2 : Rat E18 brain tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/50000 dilution

Predicted band size: 158 kDa
Observed band size: 175/240 kDa why is the actual band size different from the predicted?

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

DCC is a glycoprotein. The molecular weight observed is consistent with what have been described in the literature (PMID: 8044801).

Samples are non-boiled as boiling may cause protein aggregates.

Exposure time: 26 seconds.

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling DCC with ab273570 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on human cerebrum (PMID: 11684721). The section was incubated with ab273570 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 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat E14.5 embryo tissue labeling DCC with ab273570 at 1/100 dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (Green). Positive staining on rat embryonic brain 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).

DCC was immunoprecipitated from 0.35 mg Mouse P5 cerebral cortex tissue lysate  with ab273570 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab273570 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Mouse P5 cerebral cortex tissue lysate 10 ug

Lane 2: ab273570 IP in Mouse P5 cerebral cortex tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab273570 in Mouse P5 cerebral cortex tissue lysate

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

Exposure time: 32 seconds.

 

Flow cytometric analysis of PC-12 (Rat adrenal gland pheochromocytoma, Left) / Rat primary neuron cells (Right) cells labelling DCC with ab273570 at 1/50 dilution (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: PC12 (PMID: 8044801).

Gated on viable cells.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse E14.5 embryo tissue labeling DCC with ab273570 at 1/100 dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (Green). Positive staining on mouse embryonic brain 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).

DCC was immunoprecipitated from 0.35 mg Human hippocampus tissue lysate with ab273570 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab273570 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Human hippocampus tissue lysate 10 ug

Lane 2: ab273570 IP in Human hippocampus tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab273570 in human hippocampus tissue lysate

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

Exposure time: 10 seconds.

 

Flow cytometric analysis of Rat primary neuron cells cells labelling DCC with ab273570 at 1/50 dilution (1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Black). 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 paraffin-embedded Rat E14.5 tissue labeling DCC with ab273570 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on rat E14.5 cerebrum (PMID: 24739528). The section was incubated with ab273570 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.