a1at polymeric forms  (Hycult Biotech)

Journal: PLoS ONE

Article Title: How Can We Improve the Detection of Alpha1-Antitrypsin Deficiency?

doi: 10.1371/journal.pone.0135316

Figure Lengend Snippet: Summary of the nephelometric measurments of A1AT concentration in 966 samples analysed using routine serum protein electrophoresis (SPE) methods.

Article Snippet: Western blots were accomplished using mouse monoclonal anti-A1AT (1:500; B9, sc-59438, SantaCruz, USA), mouse monoclonal antibody, ATZ11, which recognizes both polymerized and elastase-complexed forms of A1AT (1:100, our own) and mouse monoclonal 2C1 that specifically recognizes A1AT polymeric forms (1:100, Catalog #: HM2289, Hycult Biotech, The Netherlands).

Techniques: Concentration Assay, Protein Electrophoresis

Journal: PLoS ONE

Article Title: How Can We Improve the Detection of Alpha1-Antitrypsin Deficiency?

doi: 10.1371/journal.pone.0135316

Figure Lengend Snippet: Thresholds for suspicion of severe or intermediate A1ATD according to SPE.

Article Snippet: Western blots were accomplished using mouse monoclonal anti-A1AT (1:500; B9, sc-59438, SantaCruz, USA), mouse monoclonal antibody, ATZ11, which recognizes both polymerized and elastase-complexed forms of A1AT (1:100, our own) and mouse monoclonal 2C1 that specifically recognizes A1AT polymeric forms (1:100, Catalog #: HM2289, Hycult Biotech, The Netherlands).

Techniques:

Journal: PLoS ONE

Article Title: How Can We Improve the Detection of Alpha1-Antitrypsin Deficiency?

doi: 10.1371/journal.pone.0135316

Figure Lengend Snippet: The serum concentration of A1AT was determined using routine nephelometric analysis. Equal volumes of diluted serum samples (1:100 in sterile 0.15M NaCl) were loaded and separated on the 7.5% PAGE, and Western blots were performed using specific anti-A1AT antibodies: a) mouse monoclonal against human A1AT; b) mouse monoclonal antibody, ATZ11, recognizing Z-type polymers of A1AT and c) mouse monoclonal 2C1 specific against A1AT polymers. Relative A1AI concentration in the loaded sample per well is indicated at the bottom of the gels.

Article Snippet: Western blots were accomplished using mouse monoclonal anti-A1AT (1:500; B9, sc-59438, SantaCruz, USA), mouse monoclonal antibody, ATZ11, which recognizes both polymerized and elastase-complexed forms of A1AT (1:100, our own) and mouse monoclonal 2C1 that specifically recognizes A1AT polymeric forms (1:100, Catalog #: HM2289, Hycult Biotech, The Netherlands).

Techniques: Concentration Assay, Western Blot

Journal: PLoS ONE

Article Title: How Can We Improve the Detection of Alpha1-Antitrypsin Deficiency?

doi: 10.1371/journal.pone.0135316

Figure Lengend Snippet: Serum concentrations of A1AT were determined using routine nephelometric analysis. Equal volumes of diluted serum samples (1:100 in sterile 0.15M NaCl) were loaded and separated on the 7.5% SDS-PAGE (without denaturing agents), and Western blots were performed using specific anti-A1AT antibodies: a) mouse monoclonal against human A1AT; b) mouse monoclonal antibody, ATZ11, recognizing Z-type polymers of A1AT and c) mouse monoclonal 2C1 specific against A1AT polymers. Relative A1AI concentration per well in the loaded serum sample is indicated at the bottom of the gels.

Article Snippet: Western blots were accomplished using mouse monoclonal anti-A1AT (1:500; B9, sc-59438, SantaCruz, USA), mouse monoclonal antibody, ATZ11, which recognizes both polymerized and elastase-complexed forms of A1AT (1:100, our own) and mouse monoclonal 2C1 that specifically recognizes A1AT polymeric forms (1:100, Catalog #: HM2289, Hycult Biotech, The Netherlands).

Techniques: SDS Page, Western Blot, Concentration Assay

Journal: PLoS ONE

Article Title: How Can We Improve the Detection of Alpha1-Antitrypsin Deficiency?

doi: 10.1371/journal.pone.0135316

Figure Lengend Snippet: a . Purified plasma A1AT (2.5 mg/ml) was heated at 60°C and aliquots were removed at indicated time points for analysis by a non-denaturing PAGE followed by Western blotting using rabbit polyclonal anti-A1AT antibody. b . For a more detailed analysis of the heat-treated A1AT a 4–15% gradient SDS-PAGE was used. This method is designed for separation of high molecular weight polymers. Electrophoretic separation of A1AT was followed by Western blot analysis with an anti-human A1AT antibody as above. The immunoblot patterns indicate that heat-treated A1AT typically comprises a mixture of a monomer and various sized polymers. The figure shows representative blots from 5 experiments. c . M A1AT heat-treated for 5 min exhibited a typical five band pattern in the IEF gel (lane 1). When M A1AT was heated for 15 min or longer t, the three prominent bands towards the anode were no longer detectable (lanes 2–9). Instead, a continuous smeared band occurred at the cathode (lane 3), these results paralleled those with heat-induced A1AT polymerization over time (Fig 3b). d . Initial concentrations of purified A1AT were 2.35 ± 0.042 mg/ml; n = 3. The same A1AT protein heated for a short period yielded a higher nephelometric signal than native protein. However, heating of A1AT for 45 min or longer, resulted in a remarkable decrease in the nephelometric signal relative to the native protein.

Article Snippet: Western blots were accomplished using mouse monoclonal anti-A1AT (1:500; B9, sc-59438, SantaCruz, USA), mouse monoclonal antibody, ATZ11, which recognizes both polymerized and elastase-complexed forms of A1AT (1:100, our own) and mouse monoclonal 2C1 that specifically recognizes A1AT polymeric forms (1:100, Catalog #: HM2289, Hycult Biotech, The Netherlands).

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