Anti-PAI1 antibody [EPR17272-21] - BSA and Azide free

• IP

Lab

Immunoprecipitation - Anti-PAI1 antibody [EPR17272-21] - BSA and Azide free (AB226864)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab182973).  ab182973 at 1/30 immunoprecipitating PAI1 in SW480 (human colorectal adenocarcinoma epithelial cell) whole cell lysate. Lane 1 (input) : SW480 (human colorectal adenocarcinoma epithelial cell) whole cell lysate (10µg) Lane 2 (+) : SW480 (human colorectal adenocarcinoma epithelial cell) whole cell lysate Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab182973 in SW480 whole cell lysate For western blotting, ab182973 followed by ab131366 VeriBlot for IP (HRP) was used for detection (1/5000). Blocking/Diluting buffer and concentration : 5% NFDM/TBST.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-PAI1 antibody [EPR17272-21] - BSA and Azide free (AB226864)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methonol-permeabilized Hepa1-6 (mouse hepatoma epithelial cell line) cells labeling PAI1 with ab182973 at 1/60 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077), at 1/2000 dilution was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab182973).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PAI1 antibody [EPR17272-21] - BSA and Azide free (AB226864)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Hepa1-6 (mouse hepatoma epithelial cell line) cells labeling PAI1 with ab182973 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on Hepa1-6 cells.

The nuclear counterstain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) at 1/200 dilution (red).

-ve control : PBS only, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab182973).

• IP

Lab

Immunoprecipitation - Anti-PAI1 antibody [EPR17272-21] - BSA and Azide free (AB226864)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab182973).  ab182973 at 1/30 immunoprecipitating PAI1 in SW480 (human colorectal adenocarcinoma epithelial cell) whole cell lysate. Lane 1 (input) : Mouse placenta tissue lysate 10 μg Lane 2 (+) : Mouse placenta tissue lysate Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab182973 in mouse placenta tissue lysate For western blotting, ab182973 followed by ab131366 VeriBlot for IP (HRP) were used for detection (1/5000). Blocking/Diluting buffer and concentration : 5% NFDM/TBST.

Lanes 1 - 3:

Immunoprecipitation - Anti-PAI1 antibody [EPR17272-21] (<a href='/en-us/products/primary-antibodies/pai1-antibody-epr17272-21-ab182973'>ab182973</a>) at 1/30 dilution

Lanes 1 - 3:

Immunoprecipitation - Anti-PAI1 antibody [EPR17272-21] - BSA and Azide free (ab226864)

All lanes:

Mouse placenta tissue lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 8s

• WB

Lab

Western blot - Anti-PAI1 antibody [EPR17272-21] - BSA and Azide free (AB226864)

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

The expression level of mouse and rat PAI1 may be low in normal liver tissue (PMID : 21898503). This antibody detects no bands in rat liver.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab182973).

All lanes:

Western blot - Anti-PAI1 antibody [EPR17272-21] (<a href='/en-us/products/primary-antibodies/pai1-antibody-epr17272-21-ab182973'>ab182973</a>) at 1/1000 dilution

Lane 1:

Rat placenta tissue lysate at 20 µg

Lane 2:

Rat lung tissue lysate at 20 µg

Lane 3:

Rat liver tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 45 kDa

Observed band size: 45 kDa

false

Exposure time: 20s

• WB

Lab

Western blot - Anti-PAI1 antibody [EPR17272-21] - BSA and Azide free (AB226864)

This data was developed using the same antibody clone in a different buffer formulation (ab182973).

Lanes 1 - 3 : Merged signal (red and green). Green - ab182973 observed at 48 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab182973 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 ab182973 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 [EPR17272-21] (<a href='/en-us/products/primary-antibodies/pai1-antibody-epr17272-21-ab182973'>ab182973</a>) 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

Predicted band size: 45 kDa

Observed band size: 48 kDa

false

• WB

Lab

Western blot - Anti-PAI1 antibody [EPR17272-21] - BSA and Azide free (AB226864)

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

The expression level of mouse and rat PAI1 may be low in normal liver tissue (PMID : 21898503). This antibody detects a 37kDa extra band without BOI in mouse liver.

Exposure time :

Lane 1 : 3.25 seconds
Lane 2 : 180 seconds

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab182973).

All lanes:

Western blot - Anti-PAI1 antibody [EPR17272-21] (<a href='/en-us/products/primary-antibodies/pai1-antibody-epr17272-21-ab182973'>ab182973</a>) at 1/1000 dilution

Lane 1:

Mouse placenta tissue lysate at 20 µg

Lane 2:

Mouse liver tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 45 kDa

Observed band size: 45 kDa

false