Recombinant Anti-PAI1 antibody [EPR21850-82] (ab222754)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR21850-82] to PAI1
- Suitable for: Flow Cyt (Intra), IP, ICC/IF, WB
- Reacts with: Mouse, Rat, Human
Related conjugates and formulations
Overview
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Product name
Anti-PAI1 antibody [EPR21850-82]
See all PAI1 primary antibodies -
Description
Rabbit monoclonal [EPR21850-82] to PAI1 -
Host species
Rabbit -
Tested applications
Suitable for: Flow Cyt (Intra), IP, ICC/IF, WBmore details -
Species reactivity
Reacts with: Mouse, Rat, Human -
Immunogen
Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: HepG2, HUVEC and Hepa1-6 whole cell lysate. Human, mouse and rat placenta lysate. Human liver lysate. Rat lung lysate. Serum starved NIH/3T3 treated with TGF beta1 supernatant lysate. Serum starved NIH/3T3 treated with TGF beta1 and Brefeldin A whole cell lysate. ICC/IF: HUVEC cells. Serum starved NIH/3T3 treated with TGF beta and Brefeldin A cells. Flow Cyt (intra): Serum starved NIH/3T3 treated with TGF beta and Brefeldin A cells. IP: HepG2 whole cell lysate.
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General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol, 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR21850-82 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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Recombinant Protein
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab222754 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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Flow Cyt (Intra) |
1/60.
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IP |
1/30.
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ICC/IF |
Use a concentration of 0.1 µg/ml.
This product gave a positive signal in HUVEC (-ve: HEK293) fixed with 4% formaldehyde (10 min). |
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WB |
1/1000. Predicted molecular weight: 45 kDa.
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Notes |
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Flow Cyt (Intra)
1/60. |
IP
1/30. |
ICC/IF
Use a concentration of 0.1 µg/ml. This product gave a positive signal in HUVEC (-ve: HEK293) fixed with 4% formaldehyde (10 min). |
WB
1/1000. Predicted molecular weight: 45 kDa. |
Target
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Function
This inhibitor acts as 'bait' for tissue plasminogen activator, urokinase, and protein C. Its rapid interaction with TPA may function as a major control point in the regulation of fibrinolysis. -
Tissue specificity
Found in plasma and platelets and in endothelial, hepatoma and fibrosarcoma cells. -
Involvement in disease
Defects in SERPINE1 are the cause of plasminogen activator inhibitor-1 deficiency (PAI-1D) [MIM:613329]. It is a hematologic disorder characterized by increased bleeding after trauma, injury, or surgery. Affected females have menorrhagia. The bleeding defect is due to increased fibrinolysis of fibrin blood clots due to deficiency of plasminogen activator inhibitor-1, which inhibits tissue and urinary activators of plasminogen.
Note=High concentrations of SERPINE1 seem to contribute to the development of venous but not arterial occlusions. -
Sequence similarities
Belongs to the serpin family. -
Post-translational
modificationsInactivated by proteolytic attack of the urokinase-type (u-PA) and the tissue-type (TPA), cleaving the 369-Arg-
-Met-370 bond. -
Cellular localization
Secreted. - Information by UniProt
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Database links
- Entrez Gene: 5054 Human
- Entrez Gene: 18787 Mouse
- Entrez Gene: 24617 Rat
- Omim: 173360 Human
- SwissProt: P05121 Human
- SwissProt: P22777 Mouse
- SwissProt: P20961 Rat
- Unigene: 414795 Human
see all -
Alternative names
- Clade E antibody
- Endothelial plasminogen activator inhibitor antibody
- Nexin antibody
see all
Images
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All lanes : Anti-PAI1 antibody [EPR21850-82] (ab222754) at 1/1000 dilution
Lane 1 : HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 2 : Human liver lysate
Lane 3 : Mouse placenta lysate
Lane 4 : Rat placenta lysate
Lane 5 : Hepa1-6 (Mouse hepatoma epithelial cell line) whole cell lysate
Lane 6 : Human placenta lysate
Lane 7 : HUVEC (Human umbilical vein endothelial cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
Lanes 1-5 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Lanes 6-7 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 45 kDaBlocking/Dilution buffer: 5% NFDM/TBST.
Exposure times: Lane 1-4 and 6: 3 minutes; Lane 5: 37 seconds: Lane 7: 8 seconds.
PAI1 forms complex with its target protease, t-PA (lane 7). The molecular mass observed is consistent with what has been described in the literature (PMID 21596853).
Lanes 6 and 7 were developed with a high sensitivity ECL substrate.
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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.
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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. -
Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized NIH/3T3 cells serum-starved for 4 hours, treated with TGF-ß (10ng/ml) for 3 hours, and then with TGF-ß (10ng/ml) and BFA (300ng/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).
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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. -
All lanes : Anti-HIF-1 alpha antibody [EPR16897] (ab179483) at 1/1000 dilution
Lane 1 : Mouse placenta tissue lysate
Lane 2 : Mouse lung tissue lysate
Lane 3 : Mouse liver tissue lysate
Lane 4 : Rat placenta tissue lysate
Lane 5 : Rat lung tissue lysate
Lane 6 : Rat liver tissue lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 45 kDa
Observed band size: 45 kDa
Additional bands at: 37 kDa (possible non-specific binding)
Exposure time: 3 minutesBlocking/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. -
All lanes : Anti-PAI1 antibody [EPR21850-82] (ab222754) at 1/1000 dilution
Lane 1 : NIH/3T3 (Mouse embryonic fibroblast cell line) serum-starved for 4 hours, whole cell lysate
Lane 2 : NIH/3T3 serum-starved for 4 hours then treated with 10 ng/ml TGF ß1 (ab50036) for 3 hours, then with 10 ng/ml TGF ß1 (ab50036) and 300 ng/ml Brefeldin A (BFA) together for 18 hours, whole cell lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 45 kDa
Exposure time: 32 secondsBlocking/Dilution buffer: 5% NFDM/TBST.
The expression of PAI-1 is induced by TGF-β in the NIH/3T3 cell line (PMID 17890327). The 110 kDa band likely represents PAI-1 in complex with its target protease, t-PA (PMID 21596853).
The blot was developed with a high sensitivity ECL substrate.
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All lanes : Anti-PAI1 antibody [EPR21850-82] (ab222754) at 1/1000 dilution
Lane 1 : NIH/3T3 (Mouse embryonic fibroblast cell line) serum-starved for 18 hours, then collected the supernatant lysate
Lane 2 : NIH/3T3 serum-starved for 18 hours then treated with 10 ng/ml TGF ß1 (ab50036) for 24 hours, then collected the supernatant lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 45 kDa
Exposure time: 26 secondsBlocking/Dilution buffer: 5% NFDM/TBST.
The expression of PAI-1 is induced by TGF-β in the NIH/3T3 cell line (PMID 17890327).
The blot was developed with a high sensitivity ECL substrate.
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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.
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
Certificate of Compliance
References (4)
ab222754 has been referenced in 4 publications.
- Feng Y et al. Mechanical Loading-Driven Tumor Suppression Is Mediated by Lrp5-Dependent and Independent Mechanisms. Cancers (Basel) 13:N/A (2021). PubMed: 33450808
- Kement D et al. Neuroserpin Is Strongly Expressed in the Developing and Adult Mouse Neocortex but Its Absence Does Not Perturb Cortical Lamination and Synaptic Proteome. Front Neuroanat 15:627896 (2021). PubMed: 33708076
- Li X et al. IL35 predicts prognosis in gastric cancer and is associated with angiogenesis by altering TIMP1, PAI1 and IGFBP1. FEBS Open Bio 10:2687-2701 (2020). PubMed: 33064893
- Chen C et al. Transcriptomic study of lipopolysaccharide-induced sepsis damage in a mouse heart model. Exp Ther Med 20:3782-3790 (2020). PubMed: 32855727