α1 antitrypsin

Manufactured by Merck Group

Sourced in United States

α1-antitrypsin is a laboratory equipment product. It functions as a serine protease inhibitor, which helps regulate the activity of certain enzymes involved in various biological processes.

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Lab products found in correlation

14 protocols using α1 antitrypsin

Enzymatic Activity Assay of α1-Antitrypsin and Antithrombin

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5 μM human α1-antitrypsin (Sigma) or human antithrombin (Aclotin^®, LFB) were incubated with 50 μM annonacinone (or 0.5% DMSO in reaction buffer as control) for 20 min at 37 °C. The mixtures containing α1-antitrypsin were diluted in N-Methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Sigma) containing solution and substrate hydrolysis kinetic was triggered by addition of neutrophil elastase (Sigma) to a final volume of 100 μL containing 13 nM α1-antitrypsin, 130 nM annonacinone, 82 μM chromogenic substrate and 5 nM neutrophil elastase. The mixtures containing antithrombin were diluted in fondaparinux (Arixtra^®, GlaxoSmithKline) and CS-11(22)-FXa (Hyphen Biomed) containing solution and substrate hydrolysis kinetic was triggered by addition of factor Xa (Stago BNL) to a final volume of 100 μL containing 51 nM antithrombin, 510 nM annonacinone, 200 μM chromogenic substrate, 5 μM fondaparinux and 2 nM factor Xa.

Pautus S., Alami M., Adam F., Bernadat G., Lawrence D.A., De Carvalho A., Ferry G., Rupin A., Hamze A., Champy P., Bonneau N., Gloanec P., Peglion J.L., Brion J.D., Bianchini E.P, & Borgel D. (2016). Characterization of the Annonaceous acetogenin, annonacinone, a natural product inhibitor of plasminogen activator inhibitor-1. Scientific Reports, 6, 36462.

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Fluorescein Labeling of Human Fibrinogen

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The conjugation of highly purified human fibrinogen with fluorescein isothiocyanate (FITC) via FITC-celite was performed as previously described^{64 (link)}. Sytox-Green was from Molecular Probes and Hoechst33342 from Life Technologies. Anti-CD42a-PE, mouse IgG clone Beb1, anti-CD62P-FITC, PAC-1-FITC, and CD16b were from BD Pharmingen. Anti Mac-1 activation-dependent epitope antibody (CBRM1/5) was from eBioscience. Anti-human defensin antibody was purchased from Hycult Biotech and anti-FDP-lysine antibody was from Abcam. Anti-mouse IgG-FITC (Sigma-Aldrich) was used as secondary antibody. Resveratrol (≥98% trans-3,4′,5-Trihydroxystilben) was purchased from Carl Roth, human antithrombin III from Enzyme Research Laboratories, human serum, taurine, phorbol 12-myristate 13-acetate (PMA), bovine thrombin, ADP, Thrombin Receptor Activator Peptide 6 (TRAP-6) and α1-antitrypsin were from Sigma-Aldrich and reduced glutathione for i.v. injection (Tationil®) was from Roche (Italy). IgG depleted human serum was from Sunnylab. RPMI 1640 medium was from Biochrom. Melagatran (for injection) was from Astrazeneca (Wedel, Germany). Recombinant staphylokinase (SAK) was kindly provided by Dr. Bernhard Schlott (Leibnitz Institute for Age Research, Jena, Germany) and was prepared as previously described^{65 (link)}.

Niemann S., Bertling A., Brodde M.F., Fender A.C., Van de Vyver H., Hussain M., Holzinger D., Reinhardt D., Peters G., Heilmann C., Löffler B, & Kehrel B.E. (2018). Panton-Valentine Leukocidin associated with S. aureus osteomyelitis activates platelets via neutrophil secretion products. Scientific Reports, 8, 2185.

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Multicolor Flow Cytometry Analysis

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Following mAbs were used for detection of surface markers: anti-CD11b APC (clone: ICRF44), CD48 BV421 (TÜ145), HLA-ABC FITC (G46-2.6), CD62L APC (DREG-56), CD66 PE (B1.1; all from BD Biosciences, CA, USA), HLA-C (DT9, Merck Millipore), HLA-ABC Alexa Fluor 647 (W6/32) and HLA-E PE-Cy7 (3D12; both from Biolegend), B7-H6 (875002), ULBP1 PerCP (170818), ULBP-3 PE (166510), ULBP-2/5/6 APC (165903; all from R&D Systems, MN, USA), CD56 PE-Cy7 (N901, Beckman Coulter, IN, USA), MICA/B viobright FITC (6D4, Miltenyi Biotech, Bergisch Gladbach, Germany), and Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen, CA, USA). Anti-B7-H6 (17B1.3) was kindly provided by Prof E Vivier (CMIL, Marseille, France), and anti-PVR (CD155; M5A10) and Nectin-2 (CD112; L14) were received from the lab of Prof. A Moretta (Genova, Italy). Goat serum and α1-antitrypsin were obtained from Sigma-Aldrich (MO, USA), CL097 from InvivoGen (San Diego, CA), GM-CSF from R&D Systems (MN, USA), anti-PR3 from Abcam (UK), and Batimastat and cathepsin G inhibitor from Calbiochem (CA, USA). In experiments where the extracellular FITC signal was quenched, a Fluorescin/Oregon green polyclonal antibody (ThermoFisher, Sweden) was used.

Bernson E., Christenson K., Pesce S., Pasanen M., Marcenaro E., Sivori S, & Thorén F.B. (2019). Downregulation of HLA Class I Renders Inflammatory Neutrophils More Susceptible to NK Cell-Induced Apoptosis. Frontiers in Immunology, 10, 2444.

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Surfactant Protein Immunoblotting Protocol

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Cell culture media and cell culture reagents were from Life Technologies
(Carlsbad, CA). Polymyxin B, actinomycin D,
N^ω-nitro-l-arginine methyl ester
(l-NAME), α1-antitrypsin and soybean trypsin inhibitor were from
Sigma-Aldrich (St. Louis, MO). Dimethylthiourea was from Acros Organics (Morris,
NJ). SP-A Abs were from Chemicon International (catalog # AB3030) (Temecula, CA)
or kindly provided by Joanna Floros, Penn State College of Medicine (Hershey,
PA). SP-B Abs were from Chemicon International (catalog # AB3034) (Temecula, CA)
or from Seven Hills Bioreagents (catalog # WRAB-48604) (Cincinnati, OH). SP-B
Abs from Chemicon International were reacted with the blot under reducing
conditions, whereas SP-B Abs from Seven Hills Bioreagents were reacted under
non-reducing conditions. SP-C (catalog # sc-13979) and actin (catalog # sc-1616,
sc-47778) Abs were from Santa Cruz Biotechnology (Dallas, TX) and tubulin Abs
(catalog # MS-581-P0) were from Thermo Fisher Scientific (Fremont, CA). Mouse
monoclonal SP-D Abs (catalog # WMAB-1A10A9) were from Seven Hills Bioreagents
(Cincinnati, OH). TTF-1 Abs were purified by Protein A-Sepharose chromatography
from rabbit antisera against N-terminal portion of rat T/EBP (TTF-1/Nkx2.1)
kindly provided by Shioko Kimura, National Cancer Institute (Bethesda, MD). We
have used these Abs for Western blot detection of TTF-1.^{23 (link)}

Natarajan K., Gangam K., Meganathan V., Gottipati K.R., Mitchell C, & Boggaram V. (2019). Organic dust inhibits surfactant protein expression by reducing thyroid transcription factor-1 levels in human lung epithelial cells. Innate Immunity, 25(2), 118-131.

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Blocking Surface Receptors and Cytokines

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To inhibit the function of surface receptors, cells were pretreated with dextran sulfate (100 µg/ml), heparinase III (5 U/ml), or neutralizing antibodies (5 µg/ml) directed against human TLR1, TLR2, TLR4, and TLR6 (InvivoGen) for 30 min. To suppress the biological activity of the cytokines, 10 µg/ml of antibody-neutralizing IL-6, IL-12p40, or TNF-α (BioLegend, San Diego, CA) or IL-15 (R&D Systems, Minneapolis, MN) was added. Generation of IL-32 was blocked by treating cells with α-1 antitrypsin (0.5 mg/ml; Sigma-Aldrich) (49 (link)). M-CSF receptor (M-CSFR) was blocked using GW2580 (10 μM; Abcam, Cambridge, United Kingdom) to stop M-CSF signaling. Control cells were treated with an isotype-matched antibody or an appropriate vehicle.

Wang X., Sjölinder M., Gao Y., Wan Y, & Sjölinder H. (2016). Immune Homeostatic Macrophages Programmed by the Bacterial Surface Protein NhhA Potentiate Nasopharyngeal Carriage of Neisseria meningitidis. mBio, 7(1), e01670-15.

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Enzymatic Modeling of Mitral Valve ECM

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Excised leaflets were enzymatically digested in order to model the disruption of valvular ECM components associated with MVP. Three solutions were considered: elastase only, collagenase only, and a mixture of elastase and collagenase. Porcine pancreatic elastase (Worthington Biochemical; LS006365) was dissolved into a phosphate-buffered saline (PBS) solution at a concentration of 1 unit/mL, and purified collagenase (Worthington Biochemical; LS005273) was dissolved into a PBS solution at a concentration of 64 units/mL [22 (link)]. The elastase-collagenase mixture consisted of 1 unit of elastase and 64 units of collagenase dissolved per mL of PBS. Leaflets were submerged in digestion solution and maintained at 37°C and 5% CO₂ for up to 3 hours. Stereo microscope images were recorded every 15 min for the first hour and every 30 min for the final two hours. Custom image processing analyses were used to semi-automatically identify leaflet boundaries and areas over time. Immediately following enzymatic digestion, leaflets were washed in PBS and placed in a solution of α₁-Antitrypsin (Sigma-Aldrich; A6150) at a concentration of 0.5 mg/ml for 45 minutes to arrest enzyme activity and prevent further digestion [23 (link), 24 (link)].

Bender J.M., Adams W.R., Mahadevan-Jansen A., Merryman W.D, & Bersi M.R. (2020). Radiofrequency ablation alters the microstructural organization of healthy and enzymatically digested porcine mitral valves. Experimental mechanics, 61(1), 235-251.

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Plasma-Derived α-1 Antitrypsin Purification

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α-1 Antitrypsin isolated from human plasma by chromatography was purchased from Sigma-Aldrich (A9024).

Sarabhai T., Peter C., Bär A.K., Windolf J., Relja B., Wesselborg S., Wahlers T, & Paunel-Görgülü A. (2017). Serum α-1 Antitrypsin (AAT) antagonizes intrinsic apoptosis induction in neutrophils from patients with systemic inflammatory response syndrome. PLoS ONE, 12(5), e0177450.

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Preparation of Redox-Active Compounds

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Capsaicin and A967079 were purchased from HelloBio (Bristol, UK), BCTC was purchased from Tocris (Wiesbaden-Nordenstadt, Germany). Ruthenium red, α1-antitrypsin, protoporphyrin-IX, hemin, iron(II) sulfide, sodium dithionite, DL-dithiothreitol, chelerythrine, reduced glutathione, and carvacrol were purchased from Sigma-Aldrich (Taufkirchen, Germany). hemin was resuspended in 30 mM KOH to produce a 10 mM stock solution which was stored on ice. The dilutions needed for the measurements were freshly prepared before use and dissolved in external solution.

Palmaers N.E., Wiegand S.B., Herzog C., Echtermeyer F.G., Eberhardt M.J, & Leffler A. (2021). Distinct Mechanisms Account for In Vitro Activation and Sensitization of TRPV1 by the Porphyrin Hemin. International Journal of Molecular Sciences, 22(19), 10856.

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Quantitative α-1-Antitrypsin ELISA

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The α-1-antitrypsin ELISA employed a commercial polyclonal rabbit anti-human α-1-antitrypsin primary antibody (DAKO, Cambridgeshire, UK) and a biotinylated affinity-purified rabbit anti-human α-1-antitrypsin secondary antibody. This was purified from rabbit antiserum (Dade Behring, Marburg, Germany) using antitrypsin coupled to activated CH-Sepharose 4B beads (Amersham Bioscience, Pittsburgh, USA) and conjugated with biotin using Pierce EZ-link NHS-LC Biotin^® (Pierce, Rockford, Illinois, USA). α-1-antitrypsin standards were prepared in RPMI medium plus 10% foetal calf serum, by serial 1:2 dilutions from the top standard (200 ng/ml; prepared from human plasma-purified α-1-antitrypsin, Sigma-Aldrich). ExtrAvidin^® alkaline phosphatase enzyme (Sigma-Aldrich) and phospho-nitrophenylphosphate substrate (Sigma-Aldrich) were used as a detection system. α-1-antitrypsin levels were measured using an Anthos HTII Plate reader (Anthos, UK) at an absorbance at 405 nm and quantified using GraphPad Prism software (version 5 for Mac OS X, GraphPad Software Inc.). The lower limit of detection for this assay was 0.32 ng/ml.

Dimeloe S., Rice L.V., Chen H., Cheadle C., Raynes J., Pfeffer P., Lavender P., Richards D.F., Nyon M.P., McDonnell J.M., Kemper C., Gooptu B, & Hawrylowicz C.M. (2019). Vitamin D (1,25(OH)2D3) induces α-1-antitrypsin synthesis by CD4+ T cells, which is required for 1,25(OH)2D3-driven IL-10. The Journal of Steroid Biochemistry and Molecular Biology, 189, 1-9.

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Mast Cell Purification and Tryptase Analysis

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Primary mast cells were purified from human newborn foreskin (N = 101) using anti-human c-Kit microbeads (Miltenyi Biotec) (Babina et al., 2016 (link)). Non-mast cell fractions were used for genomic DNA isolation and tryptase genotyping. Tryptase activity was assessed from mast cell lysates using Z-Gly-Pro-Arg-pNA (Bachem) in the presence of α1-antitrypsin (Sigma-Aldrich) to suppress other trypsin-like proteases. Tryptase activity is expressed as a function of equivalent concentration of active tryptase per mast cell.

Maun H.R., Jackman J.K., Choy D.F., Loyet K.M., Staton T.L., Jia G., Dressen A., Hackney J.A., Bremer M., Walters B.T., Vij R., Chen X., Trivedi N.N., Morando A., Lipari M.T., Franke Y., Wu X., Zhang J., Liu J., Wu P., Chang D., Orozco L.D., Christensen E., Wong M., Corpuz R., Hang J.Q., Lutman J., Sukumaran S., Wu Y., Ubhayakar S., Liang X., Schwartz L.B., Babina M., Woodruff P.G., Fahy J.V., Ahuja R., Caughey G.H., Kusi A., Dennis M.S., Eigenbrot C., Kirchhofer D., Austin C.D., Wu L.C., Koerber J.T., Lee W.P., Yaspan B.L., Alatsis K.R., Arron J.R., Lazarus R.A, & Yi T. (2019). An allosteric anti-tryptase antibody for the treatment of mast cell-mediated severe asthma. Cell, 179(2), 417-431.e19.

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