Recombinant Anti-PDGFR alpha + PDGFR beta antibody [Y92] - Low endotoxin, Azide free (ab215978)

All lanes : Anti-PDGFR alpha + PDGFR beta antibody [Y92] - C-terminal (ab32570) at 1/1000 dilution

Lane 1 : Recombinant human PDGFR beta protein (ab60833)
Lane 2 : Recombinant human PDGFR alpha protein (ab84797)

Lysates/proteins at 0.1 µg per lane.

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32570).

Blocking/Diluting buffer and concentration: 5% NFDM/TBST

Exposure time: 3 seconds

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

Lane 1 : Wild-type SH-SY5Y cell lysate
Lane 2 : PDGFR beta 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.

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

This data was developed using the same antibody clone in a different buffer formulation (ab32570).

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.

ab32570 staining PDGFR alpha + 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32570).

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32570).

ab32570 (purified) at 1/20 immunoprecipitating PDGFR alpha + 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32570).

ab32570 staining PDGFR alpha + 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32570).

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32570).

Intracellular Flow Cytometry analysis of NIH/3T3 (Mouse embryo fibroblast cell line) cells labeling PDGFR alpha +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. 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32570). 

 

 

Clone Y92 (ab215978) has been successfully conjugated by Abcam. This image was generated using Anti-PDGFR beta antibody [Y92] (Alexa Fluor® 488). Please refer to ab196376 for protocol details.

ab196376 staining PDGFR alpha + beta in NIH3T3 cells. The cells were fixed with 100% methanol (5 min), permeabilised in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab196376 at 1/50 dilution (shown in green) and ab195889, Mouse monoclonal [DM1A] to alpha Tubulin (Alexa Fluor® 594, shown in red) at 1/167 dilution overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32570).