Anti-PAI1 antibody [EPR21850-82] - BSA and Azide free

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-PAI1 antibody [EPR21850-82] - BSA and Azide free (AB237780)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab222754).

Flow cytometry overlay histogram showing left HUVEC positive cells and right negative HEK293 stained with ab222754 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab222754) (1x 106 in 100μl at 0.2μg/ml (1/9900)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in HUVEC Fixed with 4% formaldehyde (10 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PAI1 antibody [EPR21850-82] - BSA and Azide free (AB237780)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling PAI1 with ab222754 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1,000 dilution (green). Confocal image showing the signal is increased in 4 hour serum-starved NIH/3T3 cells treated with TGF-β (10 ng/ml) for 3 hours, then with TGF-β (10 ng/ml) and BFA (300 ng/ml) together for 18 hours. The expression of PAI-1 is induced by TGF-β in the NIH/3T3 cell line (PMID 17890327). The nuclear counter stain is DAPI (blue).
Counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at a 1/200 dilution (red).
The negative control is the secondary antibody only.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab222754).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PAI1 antibody [EPR21850-82] - BSA and Azide free (AB237780)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HUVEC (Human umbilical vein endothelial cell line) cells labeling PAI1 with ab222754 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1,000 dilution (green). Confocal image showing cytoplasmic staining in HUVEC cell line. The nuclear counter stain is DAPI (blue).
Counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at a 1/200 dilution (red).
The negative control is the secondary antibody only.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol and sodium azide (ab222754).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-PAI1 antibody [EPR21850-82] - BSA and Azide free (AB237780)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized NIH/3T3 cells serum-starved for 4 hours, treated with TGF-ß (10 ng/ml) for 3 hours, and then with TGF-ß (10 ng/ml) and BFA (300 ng/ml) together for 18 hours (Red) / Untreated control (Green) labeling PAI1 with ab222754 at 1/60 (red/green) compared with a Rabbit monoclonal IgG (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

The expression of PAI-1 is induced by TGF-ß in NIH/3T3 cell line (PMID 17890327).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab222754).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PAI1 antibody [EPR21850-82] - BSA and Azide free (AB237780)

ab222754 staining SERPINE1 in HUV-EC cells, with negative expression in HEK293 cells. The cells were fixed with 4% formaldehyde (10 min), permeabilised 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 overnight at +4°C with ab222754 at 0.1 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 0.5 μg/ml. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150119, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 647), pre-adsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab222754).

• IP

Unknown

Immunoprecipitation - Anti-PAI1 antibody [EPR21850-82] - BSA and Azide free (AB237780)

PAI1 was immunoprecipitated from 0.35 mg HepG2 (human hepatocellular carcinoma epithelial cell line) whole cell lysate with ab222754 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab222754 at 1/1,000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5,000 dilution.
Lane 1 : HepG2 whole cell lysate 10 μg (Input).
Lane 2 : ab222754 IP in HepG2 whole cell lysate (+).
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab222754 in HepG2 whole cell lysate (-).
Blocking/Dilution buffer : 5% NFDM/TBST.
Exposure time : 3 minutes.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab222754).

All lanes:

Immunoprecipitation - Anti-PAI1 antibody [EPR21850-82] (<a href='/en-us/products/primary-antibodies/pai1-antibody-epr21850-82-ab222754'>ab222754</a>)

Predicted band size: 45 kDa

false

• WB

Lab

Western blot - Anti-PAI1 antibody [EPR21850-82] - BSA and Azide free (AB237780)

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

The expression levels of mouse and rat PAI1 may be low in normal liver tissue (PMID : 21898503). This antibody detects a 37 kDa extra band and no specific band in mouse liver and no bands in rat liver.
Although lung tissue is reported to be PAI1 positive (PMID : 21768189, PMID : 17032919), this antibody can't detect band of target in mouse lung and detects weak target band in rat lung.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab179483).

All lanes:

Western blot - Anti-PAI1 antibody [EPR21850-82] (<a href='/en-us/products/primary-antibodies/pai1-antibody-epr21850-82-ab222754'>ab222754</a>) at 1/1000 dilution

Lane 1:

Mouse placenta tissue lysate at 20 µg

Lane 2:

Mouse lung tissue lysate at 20 µg

Lane 3:

Mouse liver tissue lysate at 20 µg

Lane 4:

Rat placenta tissue lysate at 20 µg

Lane 5:

Rat lung tissue lysate at 20 µg

Lane 6:

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: 37 kDa,45 kDa

false

Exposure time: 3min