All lanes : Anti-PBR antibody [EPR5384] (ab109497) at 1/1000 dilution

Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : TSPO knockout HCT116 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 19 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab109497 observed at 17 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab109497 was shown to react with PBR in wild-type HCT116 cells in western blot. Loss of signal was observed when knockout cell line ab266878 (knockout cell lysate ab257067) was used. Wild-type HCT116 and TSPO knockout HCT116 cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab109497 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human bladder carcinoma tissue labelling PBR with purified ab109497 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunohistochemistry (Frozen sections) analysis of mouse kidney tissue sections labeling PBR with Purified ab109497 at 1/250 (3.8 µg/ml).Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. DAPI was used as a counterstain.

All lanes : Anti-PBR antibody [EPR5384] (ab109497) at 1/10000 dilution

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : PBR knockout HAP1 cell lysate
Lane 3 : HEK293 cell lysate
Lane 4 : A431 cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 19 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab109497 observed at 15 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab109497 was shown to specifically react with PBR in wild type HAP1 cells. No band was observed when PBR knockout samples were used. Wild-type and PBR knockout samples were subjected to SDS-PAGE.  Ab109497 and ab8245 (loading control to GAPDH) were diluted at 1/10000 and 1/10000 dilution respectively and incubated overnight at 4C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-PBR antibody [EPR5384] (ab109497) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : TSPO knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 19 kDa
Observed band size: 15 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab109497 observed at 15 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab109497 was shown to react with PBR in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264942 (knockout cell lysate ab257066) was used. Wild-type HeLa and TSPO knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab109497 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (formalin/PFA-fixed paraffin-embedded sections) analysis of mouse hypothalamus tissue labelling PBR with ab109497 at 1/1000 dilution (1.03 μg/ml). Heat mediated antigen retrieval was performed using Tris/EDTA buffer, pH 9.0 (ab93684). A goat anti-rabbit IgG (H+L) (HRP) was used as the secondary antibody (concentration: ready to use). A secondary-antibody-only control was also performed. Counterstained with hematoxylin.

Positive staining was observed on ependymal cells and endothelial cells in mouse hypothalamus.

ab109497 staining PBR in HeLa. The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab109497 at 1μg/ml and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Flow Cytometry analysis of U-87MG cells labelling PBR with purified ab109497 at 1/150 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

ab109497 (purified) at 1/60 immunoprecipitating PBR in A431 whole cell lysate.

Lane 1 (input): A431 whole cell lysate (10µg)

Lane 2 (+): ab109497 + A431 whole cell lysate (10µg).

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab109497 in A431 whole cell lysate.

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1500 dilution.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Immunohistochemistry (Frozen sections) analysis of mouse adrenal gland tissue sections labeling PBR with Purified ab109497 at 1/250 (3.8 µg/ml).Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. DAPI was used as a counterstain.

TSPO (PBR) expression was abolished in global KO mice without pathological changes

TSPO expression in different tissues from WT and KO mice was detected by western blotting (A) and IHC (B). Scale Bars, 100μm.

4% PFA-fixed tissue sections were blocked with 5% goat serum and incubated overnight at 4°C with ab109497 at 1/1500 dilution. DAB staining.

Note: TSPO and PBR are alternative names for the same target.

(Adapted from Figure 3 of Wang et al)

TSPO (PBR) expression is localized to the mitochondria

(A) Panel shows confocal images of TSPO (red), VDAC1 (green) and nuclear counterstain (blue) in MA-10 Leydig (mouse). cells. (B) Negative control panel. Colocalization of TSPO to the mitochondrial protein VDAC1 validates the specific localization of TSPO. Scale bar 20 µm.

Cells were fixed with 4% formaldehyde and permeabilized using 0.1% Triton X-100. Cells were then blocked using 5% normal goat serum and incubated with ab109497 at 1/200 dilution.

Note: TSPO and PBR are alternative names for the same target.

ab109497 staining PBR in wild-type HAP1 cells (top panel) and PBR knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab109497 at 1μg/ml and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Immunocytochemistry/Immunofluorescence analysis of U-87MG cells labelling PBR with purified ab109497 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) were also used.

Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

All lanes : Anti-PBR antibody [EPR5384] (ab109497) at 1/50000 dilution (purified)

Lane 1 : RAW264.7 cell lysate
Lane 2 : NIH/3T3 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 19 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes : Anti-PBR antibody [EPR5384] (ab109497) at 1/50000 dilution (purified)

Lane 1 : U-87MG cell lysate
Lane 2 : HEK293 cell lysate
Lane 3 : A431 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 19 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

ab109497 (purified) at 1/60 immunoprecipitating PBR in U-87MG whole cell lysate.

Lane 1 (input): U-87MG whole cell lysate (10µg)

Lane 2 (+): ab109497 + U-87MG whole cell lysate (10µg).

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab109497 in U87-MG whole cell lysate.

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1500 dilution.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon tissue labelling PBR with unpurified ab109497 at a dilution of 1/100.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Overlay histogram showing HepG2 cells stained with unpurified ab109497 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (unpurified ab109497, 1/50 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.