Anti-PAI1 antibody [EPR17796]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PAI1 antibody [EPR17796] (AB187263)

Immunofluorescent analysis of 4% paraformaldehyde-fixed 0.1% Triton X-100 permeabilized HT1080 (Human fibrosarcoma cells) cells labeling PAI1 with ab187263 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 : ab187263 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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PAI1 antibody [EPR17796] (AB187263)

Immunofluorescent analysis of 4% paraformaldehyde-fixed 0.1% Triton X-100 permeabilized HepG2 (Human liver hepatocellular carcinoma) cells labeling PAI1 with ab187263 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 : ab187263 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.

• IP

Supplier Data

Immunoprecipitation - Anti-PAI1 antibody [EPR17796] (AB187263)

PAI1 was immunoprecipitated from 1mg of HepG2 (Human liver hepatocellular carcinoma) whole cell lysate with ab187263 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab187263 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 : ab187263 IP in HepG2 whole cell lysate. Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab187263 in HepG2 whole cell lysate.
Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 8 seconds.

All lanes:

Immunoprecipitation - Anti-PAI1 antibody [EPR17796] (ab187263)

Predicted band size: 45 kDa,62 kDa

false

• WB

Supplier Data

Western blot - Anti-PAI1 antibody [EPR17796] (AB187263)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-PAI1 antibody [EPR17796] (ab187263) at 1/10000 dilution

All lanes:

HepG2 (Human liver hepatocellular carcinoma) whole cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 45 kDa

Observed band size: 45 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-PAI1 antibody [EPR17796] (AB187263)

Lanes 1 - 4 : Merged signal (red and green). Green - ab187263 observed at 48 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab187263 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 ab187263 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.

All lanes:

Western blot - Anti-PAI1 antibody [EPR17796] (ab187263) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

SERPINE1 knockout A549 cell lysate at 20 µg

Lane 3:

HUVEC cell lysate at 20 µg

Lane 4:

HEK-293 cell lysate at 20 µg

Predicted band size: 45 kDa

Observed band size: 48 kDa

false

• WB

Supplier Data

Western blot - Anti-PAI1 antibody [EPR17796] (AB187263)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-PAI1 antibody [EPR17796] (ab187263) at 1/1000 dilution

Lane 1:

Human fetal liver lysate at 10 µg

Lane 2:

Human fetal spleen lysate at 10 µg

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

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-PAI1 antibody [EPR17796] (AB187263)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-PAI1 antibody [EPR17796] (ab187263) at 1/5000 dilution

All lanes:

Human PAI1 full length protein at 0.01 µg

Secondary

All lanes:

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

Predicted band size: 45 kDa

Observed band size: 70 kDa

false

Exposure time: 30s