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human adamts13 quantikine enzyme linked immunosorbent assay kit (R&D Systems)

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R&D Systems human adamts13 quantikine enzyme linked immunosorbent assay kit

<t>ADAMTS13</t> degradation by purified proteases and in plasma-based assays. (A) Commercial rADAMTS13 (200 nM) was incubated with various proteases (100 nM) for 2 hours at 37°C. Samples were separated under reducing conditions using SDS-PAGE, and cleavage was visualized using SYPRO Ruby. (B) rADAMTS13, varying dilutions of tissue factor (TF), and 1 mg/mL GPRP-amide were incubated in human platelet-poor plasma for 15 minutes. Thrombin generation was then initiated with calcium and quantified over time in a parallel plate by measuring fluorescence (λ ex = 360 nm; λ em = 460) at 37°C. Curves were generated using the Technothrombin TGA Software in triplicate, and representative images are shown. (C) rADAMTS13 and varying concentrations of tPA were added to human platelet-poor plasma, followed by TF and calcium. Clot formation and lysis was quantified by measuring OD at 405 nm every 30 seconds in triplicate, and representative images are shown. Samples were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and ADAMTS13 degradation was visualized via western blot using an anti-ADAMTS13 metalloprotease domain antibody. Molecular weight references are indicated on the left of blots (kDa), and full-length bands are indicated by the black arrows. APC, activated protein C; GPRP-amide, Gly-Pro-Arg-Pro-amide; OD, optical density.

Human Adamts13 Quantikine Enzyme Linked Immunosorbent Assay Kit, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/human+adamts13+quantikine+enzyme+linked+immunosorbent+assay+kit/Recombinant+Human+ADAMTS13+Protein%2C+CF/pmc12159909-40-3-10
Average 94 stars, based on 35 article reviews

human adamts13 quantikine enzyme linked immunosorbent assay kit - by Bioz Stars, 2026-10

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1) Product Images from "Development of a protease-resistant ADAMTS13 to improve stability against proteolytic degradation ∗ "

Article Title: Development of a protease-resistant ADAMTS13 to improve stability against proteolytic degradation ∗

Journal: Blood Advances

doi: 10.1182/bloodadvances.2024015212

ADAMTS13 degradation by purified proteases and in plasma-based assays. (A) Commercial rADAMTS13 (200 nM) was incubated with various proteases (100 nM) for 2 hours at 37°C. Samples were separated under reducing conditions using SDS-PAGE, and cleavage was visualized using SYPRO Ruby. (B) rADAMTS13, varying dilutions of tissue factor (TF), and 1 mg/mL GPRP-amide were incubated in human platelet-poor plasma for 15 minutes. Thrombin generation was then initiated with calcium and quantified over time in a parallel plate by measuring fluorescence (λ ex = 360 nm; λ em = 460) at 37°C. Curves were generated using the Technothrombin TGA Software in triplicate, and representative images are shown. (C) rADAMTS13 and varying concentrations of tPA were added to human platelet-poor plasma, followed by TF and calcium. Clot formation and lysis was quantified by measuring OD at 405 nm every 30 seconds in triplicate, and representative images are shown. Samples were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and ADAMTS13 degradation was visualized via western blot using an anti-ADAMTS13 metalloprotease domain antibody. Molecular weight references are indicated on the left of blots (kDa), and full-length bands are indicated by the black arrows. APC, activated protein C; GPRP-amide, Gly-Pro-Arg-Pro-amide; OD, optical density.

Figure Legend Snippet: ADAMTS13 degradation by purified proteases and in plasma-based assays. (A) Commercial rADAMTS13 (200 nM) was incubated with various proteases (100 nM) for 2 hours at 37°C. Samples were separated under reducing conditions using SDS-PAGE, and cleavage was visualized using SYPRO Ruby. (B) rADAMTS13, varying dilutions of tissue factor (TF), and 1 mg/mL GPRP-amide were incubated in human platelet-poor plasma for 15 minutes. Thrombin generation was then initiated with calcium and quantified over time in a parallel plate by measuring fluorescence (λ ex = 360 nm; λ em = 460) at 37°C. Curves were generated using the Technothrombin TGA Software in triplicate, and representative images are shown. (C) rADAMTS13 and varying concentrations of tPA were added to human platelet-poor plasma, followed by TF and calcium. Clot formation and lysis was quantified by measuring OD at 405 nm every 30 seconds in triplicate, and representative images are shown. Samples were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and ADAMTS13 degradation was visualized via western blot using an anti-ADAMTS13 metalloprotease domain antibody. Molecular weight references are indicated on the left of blots (kDa), and full-length bands are indicated by the black arrows. APC, activated protein C; GPRP-amide, Gly-Pro-Arg-Pro-amide; OD, optical density.

Techniques Used: Purification, Clinical Proteomics, Incubation, SDS Page, Fluorescence, Generated, Software, Lysis, Polyacrylamide Gel Electrophoresis, Western Blot, Molecular Weight

ADAMTS13 degradation by activated neutrophils. (A) ADAMTS13 was incubated with 0 × 10 3 to 500 × 10 3 PMA-stimulated neutrophils for 1 hour at 37°C, in the absence or presence of 2 mM AEBSF or 5 mM EDTA (B). (C) ADAMTS13 was incubated with 5 × 10 3 PMA-stimulated neutrophils with increasing concentrations of AEBSF, cathepsinG inhibitor I (CGI I), or Sivelestat for 1 hour. Samples were separated via SDS-PAGE and analyzed by (A) western blot using an anti-ADAMTS13 metalloprotease domain antibody, or (B-C) SYPRO Ruby. Black arrows indicate the level of full-length ADAMTS13 (∼180 kDa). AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride; EDTA, ethylenediaminetetraacetic acid.

Figure Legend Snippet: ADAMTS13 degradation by activated neutrophils. (A) ADAMTS13 was incubated with 0 × 10 3 to 500 × 10 3 PMA-stimulated neutrophils for 1 hour at 37°C, in the absence or presence of 2 mM AEBSF or 5 mM EDTA (B). (C) ADAMTS13 was incubated with 5 × 10 3 PMA-stimulated neutrophils with increasing concentrations of AEBSF, cathepsinG inhibitor I (CGI I), or Sivelestat for 1 hour. Samples were separated via SDS-PAGE and analyzed by (A) western blot using an anti-ADAMTS13 metalloprotease domain antibody, or (B-C) SYPRO Ruby. Black arrows indicate the level of full-length ADAMTS13 (∼180 kDa). AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride; EDTA, ethylenediaminetetraacetic acid.

Techniques Used: Incubation, SDS Page, Western Blot

Characterizing the resistance of the ADAMTS13 mutants against proteolysis by purified proteases and in plasma fibrinolysis. (A) Four mutants of ADAMTS13 were developed: 2 with 1 of each C-terminal linker region mutated (T4L and T8L mutants), 1 with both linker regions mutated (T4L/T8L mutant), and a fourth with both linker regions and an additional elastase cleavage site mutation (T4L/T8L[I380G] mutant). (B) Using commercial recombinant ADAMTS13 (WT) as a control, each linker mutant (T4L, T8L, and/or T4L/T8L) was incubated at 50 nM with purified coagulation and fibrinolytic proteases at 50 nM (plasmin, thrombin, and kallikrein) or 100 nM (FXIa) for 0 to 180 minutes at 37°C. (C) The T4L/T8L mutant and 10 nM tPA were added to human platelet-poor plasma, followed by tissue factor and calcium at 37°C. Aliquots were removed at 0 and 45 minutes. (D) Mutants were incubated with 50 nM purified neutrophil-derived proteases (elastase, proteinase 3, and cathepsin G) at 37°C. The T4L/T8L(I380G) mutant was also incubated with 50 nM elastase. Western blotting was performed using an anti-ADAMTS13 metalloprotease domain antibody. The band corresponding to full-length protein (∼180 kDa) is indicated by black arrows.

Figure Legend Snippet: Characterizing the resistance of the ADAMTS13 mutants against proteolysis by purified proteases and in plasma fibrinolysis. (A) Four mutants of ADAMTS13 were developed: 2 with 1 of each C-terminal linker region mutated (T4L and T8L mutants), 1 with both linker regions mutated (T4L/T8L mutant), and a fourth with both linker regions and an additional elastase cleavage site mutation (T4L/T8L[I380G] mutant). (B) Using commercial recombinant ADAMTS13 (WT) as a control, each linker mutant (T4L, T8L, and/or T4L/T8L) was incubated at 50 nM with purified coagulation and fibrinolytic proteases at 50 nM (plasmin, thrombin, and kallikrein) or 100 nM (FXIa) for 0 to 180 minutes at 37°C. (C) The T4L/T8L mutant and 10 nM tPA were added to human platelet-poor plasma, followed by tissue factor and calcium at 37°C. Aliquots were removed at 0 and 45 minutes. (D) Mutants were incubated with 50 nM purified neutrophil-derived proteases (elastase, proteinase 3, and cathepsin G) at 37°C. The T4L/T8L(I380G) mutant was also incubated with 50 nM elastase. Western blotting was performed using an anti-ADAMTS13 metalloprotease domain antibody. The band corresponding to full-length protein (∼180 kDa) is indicated by black arrows.

Techniques Used: Purification, Clinical Proteomics, Mutagenesis, Recombinant, Control, Incubation, Coagulation, Derivative Assay, Western Blot

Resistance of ADAMTS13 mutants to activated neutrophils. 50 nM WT-, T4L/T8L-, or T4L/T8L(I380G)-ADAMTS13 was incubated with 0 × 10 3 to 50 × 10 3 PMA-activated neutrophils, or 500 × 10 3 neutrophils, as indicated. Reactions took place for 1 hour at 37°C, then samples were removed and separated via SDS-PAGE under reducing conditions. Cleavage was visualized by western blot using an anti-ADAMTS13 metalloprotease domain antibody. Molecular weight references are indicated to the left (kDa). The level of full-length protein (∼180 kDa) is indicated by black arrows.

Figure Legend Snippet: Resistance of ADAMTS13 mutants to activated neutrophils. 50 nM WT-, T4L/T8L-, or T4L/T8L(I380G)-ADAMTS13 was incubated with 0 × 10 3 to 50 × 10 3 PMA-activated neutrophils, or 500 × 10 3 neutrophils, as indicated. Reactions took place for 1 hour at 37°C, then samples were removed and separated via SDS-PAGE under reducing conditions. Cleavage was visualized by western blot using an anti-ADAMTS13 metalloprotease domain antibody. Molecular weight references are indicated to the left (kDa). The level of full-length protein (∼180 kDa) is indicated by black arrows.

Techniques Used: Incubation, SDS Page, Western Blot, Molecular Weight

Activity of ADAMTS13, MDTCS, and T4L/T8L-ADAMTS13 in a microfluidic flow assay. DiOC6-stained platelets were perfused over a microfluidic flow chamber lined with histamine-stimulated HUVECs, forming stable VWF-platelet strings. Channels were then perfused with 5 mL of 5 nM MDTCS, 5 nM WT-ADAMTS13, 5 nM T4L/T8L-ADAMTS13, or ADAMTS13 reaction buffer (n = 3). Using a Leica Stellaris 5 inverted confocal microscope (10× dry lens; λ ex = 490 nm; λ em = 525 nm), videos were captured at 0.77 frames per second; (A) photographic representation of experimental replicate. Experiments occurred at room temperature. The total length of strings within the frame was quantified every 20 frames for 5 frames. Rates were determined using linear regression (B), and differences between rates were assessed using unpaired t tests . Error bars represent standard deviation, and symbols represent 4 experimental conditions: buffer (▲), MDTCS (■), WT-ADAMTS13 (●), T4L/T8L-ADAMTS13 (▼). ∗P < .05, vs MDTCS.

Figure Legend Snippet: Activity of ADAMTS13, MDTCS, and T4L/T8L-ADAMTS13 in a microfluidic flow assay. DiOC6-stained platelets were perfused over a microfluidic flow chamber lined with histamine-stimulated HUVECs, forming stable VWF-platelet strings. Channels were then perfused with 5 mL of 5 nM MDTCS, 5 nM WT-ADAMTS13, 5 nM T4L/T8L-ADAMTS13, or ADAMTS13 reaction buffer (n = 3). Using a Leica Stellaris 5 inverted confocal microscope (10× dry lens; λ ex = 490 nm; λ em = 525 nm), videos were captured at 0.77 frames per second; (A) photographic representation of experimental replicate. Experiments occurred at room temperature. The total length of strings within the frame was quantified every 20 frames for 5 frames. Rates were determined using linear regression (B), and differences between rates were assessed using unpaired t tests . Error bars represent standard deviation, and symbols represent 4 experimental conditions: buffer (▲), MDTCS (■), WT-ADAMTS13 (●), T4L/T8L-ADAMTS13 (▼). ∗P < .05, vs MDTCS.

Techniques Used: Activity Assay, Staining, Microscopy, Standard Deviation

Recombinant:

Article Title: Development of a protease-resistant ADAMTS13 to improve stability against proteolytic degradation
Article Snippet: .. Recombinant (r)ADAMTS13 and human ADAMTS13 Quantikine enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN), kallikrein, FXIIa (Enzyme Research Laboratories, South Bend, IN), thrombin, FXa, FIXa, FXIa, FVIIa, activated protein C (APC), elastase, cathepsin G (Sigma-Aldrich, St Louis, MO), proteinase 3 (Enzo Life Sciences, Inc, Farmingdale, NY), FRETS-VWF73 (AnaSpec, Fremont, CA), AEBSF (4-[2-aminoethyl]benzenesulfonyl fluoride hydrochloride), Sivelestat (Tocris Bioscience, Bristol, United Kingdom), and cathepsin G inhibitor 1 (Cayman Chemical Company, Ann Arbor, MI). ..

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Journal: Blood Advances

Article Title: Development of a protease-resistant ADAMTS13 to improve stability against proteolytic degradation ∗

doi: 10.1182/bloodadvances.2024015212

Figure Lengend Snippet: ADAMTS13 degradation by purified proteases and in plasma-based assays. (A) Commercial rADAMTS13 (200 nM) was incubated with various proteases (100 nM) for 2 hours at 37°C. Samples were separated under reducing conditions using SDS-PAGE, and cleavage was visualized using SYPRO Ruby. (B) rADAMTS13, varying dilutions of tissue factor (TF), and 1 mg/mL GPRP-amide were incubated in human platelet-poor plasma for 15 minutes. Thrombin generation was then initiated with calcium and quantified over time in a parallel plate by measuring fluorescence (λ ex = 360 nm; λ em = 460) at 37°C. Curves were generated using the Technothrombin TGA Software in triplicate, and representative images are shown. (C) rADAMTS13 and varying concentrations of tPA were added to human platelet-poor plasma, followed by TF and calcium. Clot formation and lysis was quantified by measuring OD at 405 nm every 30 seconds in triplicate, and representative images are shown. Samples were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and ADAMTS13 degradation was visualized via western blot using an anti-ADAMTS13 metalloprotease domain antibody. Molecular weight references are indicated on the left of blots (kDa), and full-length bands are indicated by the black arrows. APC, activated protein C; GPRP-amide, Gly-Pro-Arg-Pro-amide; OD, optical density.

Article Snippet: Recombinant (r)ADAMTS13 and human ADAMTS13 Quantikine enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN), kallikrein, FXIIa (Enzyme Research Laboratories, South Bend, IN), thrombin, FXa, FIXa, FXIa, FVIIa, activated protein C (APC), elastase, cathepsin G (Sigma-Aldrich, St Louis, MO), proteinase 3 (Enzo Life Sciences, Inc, Farmingdale, NY), FRETS-VWF73 (AnaSpec, Fremont, CA), AEBSF (4-[2-aminoethyl]benzenesulfonyl fluoride hydrochloride), Sivelestat (Tocris Bioscience, Bristol, United Kingdom), and cathepsin G inhibitor 1 (Cayman Chemical Company, Ann Arbor, MI).

Techniques: Purification, Clinical Proteomics, Incubation, SDS Page, Fluorescence, Generated, Software, Lysis, Polyacrylamide Gel Electrophoresis, Western Blot, Molecular Weight

Journal: Blood Advances

Article Title: Development of a protease-resistant ADAMTS13 to improve stability against proteolytic degradation ∗

doi: 10.1182/bloodadvances.2024015212

Figure Lengend Snippet: ADAMTS13 degradation by activated neutrophils. (A) ADAMTS13 was incubated with 0 × 10 3 to 500 × 10 3 PMA-stimulated neutrophils for 1 hour at 37°C, in the absence or presence of 2 mM AEBSF or 5 mM EDTA (B). (C) ADAMTS13 was incubated with 5 × 10 3 PMA-stimulated neutrophils with increasing concentrations of AEBSF, cathepsinG inhibitor I (CGI I), or Sivelestat for 1 hour. Samples were separated via SDS-PAGE and analyzed by (A) western blot using an anti-ADAMTS13 metalloprotease domain antibody, or (B-C) SYPRO Ruby. Black arrows indicate the level of full-length ADAMTS13 (∼180 kDa). AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride; EDTA, ethylenediaminetetraacetic acid.

Techniques: Incubation, SDS Page, Western Blot

Journal: Blood Advances

Article Title: Development of a protease-resistant ADAMTS13 to improve stability against proteolytic degradation ∗

doi: 10.1182/bloodadvances.2024015212

Figure Lengend Snippet: Characterizing the resistance of the ADAMTS13 mutants against proteolysis by purified proteases and in plasma fibrinolysis. (A) Four mutants of ADAMTS13 were developed: 2 with 1 of each C-terminal linker region mutated (T4L and T8L mutants), 1 with both linker regions mutated (T4L/T8L mutant), and a fourth with both linker regions and an additional elastase cleavage site mutation (T4L/T8L[I380G] mutant). (B) Using commercial recombinant ADAMTS13 (WT) as a control, each linker mutant (T4L, T8L, and/or T4L/T8L) was incubated at 50 nM with purified coagulation and fibrinolytic proteases at 50 nM (plasmin, thrombin, and kallikrein) or 100 nM (FXIa) for 0 to 180 minutes at 37°C. (C) The T4L/T8L mutant and 10 nM tPA were added to human platelet-poor plasma, followed by tissue factor and calcium at 37°C. Aliquots were removed at 0 and 45 minutes. (D) Mutants were incubated with 50 nM purified neutrophil-derived proteases (elastase, proteinase 3, and cathepsin G) at 37°C. The T4L/T8L(I380G) mutant was also incubated with 50 nM elastase. Western blotting was performed using an anti-ADAMTS13 metalloprotease domain antibody. The band corresponding to full-length protein (∼180 kDa) is indicated by black arrows.

Techniques: Purification, Clinical Proteomics, Mutagenesis, Recombinant, Control, Incubation, Coagulation, Derivative Assay, Western Blot

Journal: Blood Advances

Article Title: Development of a protease-resistant ADAMTS13 to improve stability against proteolytic degradation ∗

doi: 10.1182/bloodadvances.2024015212

Figure Lengend Snippet: Resistance of ADAMTS13 mutants to activated neutrophils. 50 nM WT-, T4L/T8L-, or T4L/T8L(I380G)-ADAMTS13 was incubated with 0 × 10 3 to 50 × 10 3 PMA-activated neutrophils, or 500 × 10 3 neutrophils, as indicated. Reactions took place for 1 hour at 37°C, then samples were removed and separated via SDS-PAGE under reducing conditions. Cleavage was visualized by western blot using an anti-ADAMTS13 metalloprotease domain antibody. Molecular weight references are indicated to the left (kDa). The level of full-length protein (∼180 kDa) is indicated by black arrows.

Techniques: Incubation, SDS Page, Western Blot, Molecular Weight

Journal: Blood Advances

Article Title: Development of a protease-resistant ADAMTS13 to improve stability against proteolytic degradation ∗

doi: 10.1182/bloodadvances.2024015212

Figure Lengend Snippet: Activity of ADAMTS13, MDTCS, and T4L/T8L-ADAMTS13 in a microfluidic flow assay. DiOC6-stained platelets were perfused over a microfluidic flow chamber lined with histamine-stimulated HUVECs, forming stable VWF-platelet strings. Channels were then perfused with 5 mL of 5 nM MDTCS, 5 nM WT-ADAMTS13, 5 nM T4L/T8L-ADAMTS13, or ADAMTS13 reaction buffer (n = 3). Using a Leica Stellaris 5 inverted confocal microscope (10× dry lens; λ ex = 490 nm; λ em = 525 nm), videos were captured at 0.77 frames per second; (A) photographic representation of experimental replicate. Experiments occurred at room temperature. The total length of strings within the frame was quantified every 20 frames for 5 frames. Rates were determined using linear regression (B), and differences between rates were assessed using unpaired t tests . Error bars represent standard deviation, and symbols represent 4 experimental conditions: buffer (▲), MDTCS (■), WT-ADAMTS13 (●), T4L/T8L-ADAMTS13 (▼). ∗P < .05, vs MDTCS.

Techniques: Activity Assay, Staining, Microscopy, Standard Deviation

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human adamts13 quantikine enzyme linked immunosorbent assay kit (R&D Systems)

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1) Product Images from "Development of a protease-resistant ADAMTS13 to improve stability against proteolytic degradation ∗ "

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