ab32570 staining PDGFR beta in human lung cancer tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Shows positive staining on stromal cells. Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Samples were incubated with primary antibody (1/500). An HRP-conjugated Goat anti-rabbit IgG (ready to use) was used as the secondary antibody. Counter stained with Hematoxylin.

All lanes : Anti-PDGFR beta antibody [Y92] - C-terminal (ab32570) at 1/5000 dilution

Lane 1 : Wild-type SH-SY5Y cell lysate
Lane 2 : PDGFRB knockout SH-SY5Y cell lysate
Lane 3 : Human Skeletal Muscle tissue lysate
Lane 4 : HeLa cell lysate

Lysates/proteins at 30 µg per lane.

Performed under reducing conditions.

Predicted band size: 124 kDa
Observed band size: 170 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab32570 observed at 170 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab32570 was shown to react with PDGFRB in wild-type SH-SY5Y cells in Western blot with loss of signal observed in PDGFRB knockout cell line ab273749 (knockout cell lysate ab275523). Wild-type SH-SY5Y and PDGFRB knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab32570 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 5000 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.

Immunofluorescence analysis of NIH/3T3 (Mouse embryo fibroblast cell line) cells stimulated with PDGF, staining PDGFR beta with unpurified ab32570.

Flow cytometry analysis of NIH/3T3 (Mouse embryo fibroblast cell line) cells labeling PDGFR beta (red) with ab32570 at a 1/20 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730). Blue (unlabeled control) - Cells without incubation with primary and secondary antibodies.

Immunohistochemical staining of paraffin embedded human spleen with purified ab32570 at a working dilution of 1/50. The secondary antibody used is ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L), at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

 

Immunofluorescence staining of NIH/3T3 (Mouse embryo fibroblast cell line) cells with purified ab32570 at a working dilution of 1 in 100, counter-stained with DAPI. Tubulin was stained with mouse anti-tubulin at a dilution of 1/1000 (ab7291) and Alexa Fluor^® 594 goat anti-mouse at a dilution of 1/500 (ab150120) . The secondary antibody was ab150077 Alexa Fluor^® 488 goat anti rabbit, used at a dilution of 1 in 500. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100.

The negative controls are shown in the bottom middle and right hand panels - for the first negative control, purified ab32570 was used at a dilution of 1/200 followed by an Alexa Fluor^® 555 goat anti-mouse antibody at a dilution of 1/500 and for the second negative control mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab15007) were used.

All lanes : Anti-PDGFR beta antibody [Y92] - C-terminal (ab32570) at 1/10000 dilution (purified)

Lane 1 : Rat brain tissue lysate
Lane 2 : Rat heart tissue lysate
Lane 3 : Mouse brain tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 124 kDa
Observed band size: 175 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

ab32570 (purified) at 1/20 immunoprecipitating PDGFR beta in NIH/3T3 (Mouse embryo fibroblast cell line) (Lane 1 and 2). Lane 3 - PBS.

For western blotting a HRP-conjugated anti-rabbit IgG specific to the non-reduced form of IgG was used as the secondary antibody (1/1500).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Anti-PDGFR beta antibody [Y92] - C-terminal (ab32570) at 1/10000 dilution (purified) + SH-SY5Y (Human neuroblastoma cell line from bone marrow) cell lysate at 10 µg

Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 124 kDa
Observed band size: 175 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Anti-PDGFR beta antibody [Y92] - C-terminal (ab32570) at 1/5000 dilution (purified) + Human fetal brain tissue lysate at 10 µg

Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 124 kDa
Observed band size: 175 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Anti-PDGFR beta antibody [Y92] - C-terminal (ab32570) at 1/50000 dilution (purified) + NIH/3T3 (Mouse embryo fibroblast cell line) cell lysate at 10 µg

Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 124 kDa
Observed band size: 175 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

ab32570 staining PDGFR beta in human breast tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Shows positive staining on stromal cells. Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Samples were incubated with primary antibody (1/500). An HRP-conjugated Goat anti-rabbit IgG (ready to use) was used as the secondary antibody. Counter stained with Hematoxylin.