HSA and human α-1 antitrypsin isolated from human plasma were purchased from Sigma (Saint Louis, MO, USA). Intravenous immunoglobulin (IVIG from Green Cross, Yongin, Korea) was used as pooled human immunoglobulin G (phIgG). Sterile human serum purchased from Sigma was used after heat inactivation.
Human α1 antitrypsin
Manufactured by Merck Group
Sourced in United States
Human α1-antitrypsin is a laboratory product used for research purposes. It is a protein that functions as a protease inhibitor, regulating the activity of certain enzymes in the body. This product is intended for use in scientific investigations and studies, and its core function is to serve as a research tool.
Automatically generated - may contain errors
Lab products found in correlation
Sterile human serum, by Merck Group (1 mentions)
Human serum albumin (hsa), by Merck Group (1 mentions)
Arixtra, by GlaxoSmithKline (1 mentions)
Neutrophil elastase, by Merck Group (1 mentions)
N methoxysuccinyl ala ala pro val p nitroanilide, by Merck Group (1 mentions)
α1 antitrypsin, by Merck Group (1 mentions)
Human transferrin apoform transferrin, by Fujifilm (1 mentions)
Human α1agp, by Merck Group (1 mentions)
Streptococcus pneumoniae β galactosidase, by New England Biolabs (1 mentions)
Arthrobacter ureafaciens neuraminidase, by Nacalai Tesque (1 mentions)
Human haptoglobin, by Merck Group (1 mentions)
Human fibrinogen, by Fujifilm (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Mops sds running buffer, by Thermo Fisher Scientific (1 mentions)
Anti rabbit horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
4 protocols using human α1 antitrypsin
1
Isolation and Characterization of Plasma Proteins
2
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.
3
Glycoprotein Deglycosylation Protocol
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Human transferrin apoform (transferrin) (Wako), human α1AGP (Sigma; catalog no.: G9885), human fibrinogen (Fujifilm; catalog no.: 061-03691), human haptoglobin (Sigma; catalog no.: H3536), human α1-antitrypsin (Sigma; catalog no.: A9024), and human IgG (Wako; catalog no.: 143-09501) were purchased and used for the assays. One milligram of α1AGP, transferrin, fibrinogen, haptoglobin, α1-antitrypsin, or IgG was digested with 3.8 munit/μl of Arthrobacter ureafaciens neuraminidase (Nacalai Tesque) and 1.2 unit/μl of Streptococcus pneumoniae β-galactosidase (New England Biolabs) in 87 mM sodium acetate buffer (pH 4.5) at 37 °C overnight.
4
Quantification of Urinary Proteins by Western Blot
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Pooled urinary protein samples (10 μg) were separated on 4-12 % Bis-Tris minigel in MOPS SDS running buffer (ThermoFisher Scientific, Waltham, Massachusetts, USA). Electrophoresis was performed at a constant voltage 200 V. Proteins were then transferred to PVDF membrane (GE Healthcare Life Sciences, Little Chalfont, United Kingdom) in wet Mini Blot module from ThermoFisher Scientific at a constant voltage 20 V. Membrane was blocked for one hour in 5 % milk in tris-buffered saline containing 0.1 % Tween 20, and incubated overnight with the primary antibody in 5 % milk. Rabbit primary antibodies against human uromodulin (1:6000) from BioVendor, Brno, Czech Republic and human α-1-antitrypsin (1:1000) from Sigma were used. Anti-rabbit horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, Pennsylvania, USA) was added to the membrane for one hour and a signal was detected using a chemiluminescent luminol-based substrate (Cell Signaling Technology, Danvers, Massachusetts, USA).
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