All lanes : Anti-PAI1 antibody [EPR17795] (ab187262) at 1/1000 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : SERPINE1 knockout A549 cell lysate
Lane 3 : HUVEC cell lysate
Lane 4 : HEK-293 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 45 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab187262 observed at 48 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab187262 was shown to react with PAI1 in wild-type A549 cells in Western blot. The band observed in the edited lysate lane above 45 kDa is likely to represent SERPINE1 with an insertion. This has not been investigated further. Wild-type A549 and SERPINE1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab187262 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.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HepG2 (Human liver hepatocellular carcinoma) cells labeling PAI1 with ab187262 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/400 dilution (green). Cytoplasm staining on HepG2 cells was observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).

The negative controls are as follows:
-ve control 1: ab187262 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution.

ab187262 staining PAI1 in the human cell line HepG2 (human hepatocellular carcinoma) by flow cytometry. Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/60. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control: Rabbit monoclonal IgG (Black)

Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

PAI1 was immunoprecipitated from 1mg of HepG2 (Human liver hepatocellular carcinoma) whole cell lysate with ab187262 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab187262 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1500 dilution.

Lane 1: HepG2 whole cell lysate 10 µg (Input). Lane 2: ab187262 IP in HepG2 whole cell lysate. Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab187262 in HepG2 whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 30 seconds.

All lanes : Anti-PAI1 antibody [EPR17795] (ab187262) at 1/1000 dilution

Lane 1 : Human fetal liver lysate
Lane 2 : Human fetal spleen lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 45 kDa
Observed band size: 45 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HT1080 (Human fibrosarcoma cells) cells labeling PAI1 with ab187262 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/400 dilution (green). Cytoplasm staining on HT1080 cells was observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).

The negative controls are as follows:
-ve control 1: ab187262 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution.

Anti-PAI1 antibody [EPR17795] (ab187262) at 1/5000 dilution + HepG2 (Human liver hepatocellular carcinoma) whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 45 kDa
Observed band size: 45 kDa


Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-PAI1 antibody [EPR17795] (ab187262) at 1/5000 dilution + Human PAI1 full length protein at 0.01 µg

Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 45 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.