Immunohistochemical analysis of paraffin-embedded Human colonic carcinoma tissue labeling PDGFR alpha with ab134123 at 1/500 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on stromal cells in human colonic carcinoma. The section was incubated with ab134123 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-PDGFR alpha antibody [EPR5480] (ab134123) at 1/1000 dilution

Lane 1 : Wild-type SH-SY5Y cell lysate
Lane 2 : PDGFRA knockout SH-SY5Y cell lysate

Lysates/proteins at 40 µg per lane.

Performed under reducing conditions.

Predicted band size: 122 kDa
Observed band size: 180 kDa why is the actual band size different from the predicted?

Lanes 1 - 2: Merged signal (red and green). Green - ab134123 observed at 180 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab134123 was shown to react with PDGFR alpha in wild-type SH-SY5Y cells in Western blot with loss of signal observed in PDGFRA knockout cell line ab275335 (knockout cell lysate ab275522). Wild-type SH-SY5Y and PDGFRA knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab134123 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

Flow cytometry analysis of L6(Rat skeletal muscle myoblast) cells labelling PDGFR alpha with ab134123 at 1/20 dilution (14.65 µg/ml) (Red). Cells were fixed with 4% paraformaldehyde . Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) (Black). Unlabeled control - Unlabelled cells (Blue).

All lanes : Anti-PDGFR alpha antibody [EPR5480] (ab134123) at 1/1000 dilution

Lane 1 : Human skeletal muscle lysate
Lane 2 : Human spleen lysate
Lane 3 : Human fetal brain lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat anti-rabbit HRP conjugated antibody at 1/2000 dilution

Predicted band size: 122 kDa
Observed band size: 190 kDa why is the actual band size different from the predicted?

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling PDGFR alpha with ab134123 at 1/500 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on stromal cells in human colon (PMID: 23429582). The section was incubated with ab134123 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

Flow cytometry analysis of SH-SY5Y (Human neuroblastoma epithelial cell) cells labelling PDGFR alpha with ab134123 at 1/20 dilution (14.65 µg/ml) (Red). Cells were fixed with 4% paraformaldehyde . Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) (Black). Unlabeled control - Unlabelled cells (Blue).

ELISA analysis of Human PDGFR alpha recombinant protein at 250 ng/mL with ab134123. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution was used as the secondary antibody.

All lanes : Anti-PDGFR alpha antibody [EPR5480] (ab134123) at 1/1000 dilution

Lane 1 : SH-SY5Y (human neuroblastoma cell line from bone marrow) cell lysate
Lane 2 : C2C12 (mouse myoblast cell line) cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat anti-rabbit HRP conjugated antibody at 1/2000 dilution

Predicted band size: 122 kDa
Observed band size: 190 kDa why is the actual band size different from the predicted?