a1at polymeric forms  (Hycult Biotech)


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    Structured Review

    Hycult Biotech a1at polymeric forms
    Summary of the nephelometric measurments of <t> A1AT </t> concentration in 966 samples analysed using routine serum protein electrophoresis (SPE) methods.
    A1at Polymeric Forms, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/a1at polymeric forms/product/Hycult Biotech
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    a1at polymeric forms - by Bioz Stars, 2024-06
    93/100 stars

    Images

    1) Product Images from "How Can We Improve the Detection of Alpha1-Antitrypsin Deficiency?"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0135316

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

    Techniques Used: Concentration Assay, Protein Electrophoresis

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

    Techniques Used:

    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.
    Figure Legend 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.

    Techniques Used: Concentration Assay, Western Blot

    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.
    Figure Legend 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.

    Techniques Used: SDS Page, Western Blot, Concentration Assay

    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.
    Figure Legend 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.

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

    polymeric a1at  (Hycult Biotech)


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  • 86

    Structured Review

    Hycult Biotech polymeric a1at
    FA content in different <t> A1AT </t> preparations
    Polymeric A1at, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polymeric a1at/product/Hycult Biotech
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    polymeric a1at - by Bioz Stars, 2024-06
    86/100 stars

    Images

    1) Product Images from "Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression"

    Article Title: Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1500740

    FA content in different  A1AT  preparations
    Figure Legend Snippet: FA content in different A1AT preparations

    Techniques Used: Affinity Purification

    Pull down of FA-free HSA-0 and A1AT-0 by FA-coupled agarose beads. (A) Fatty acid free HSA-0 or A1AT-0 (Zemaira) was mixed on a rotating mixer with ω-aminohexyl-agarose beads coupled to LA, OA or control (co.) beads exposed to coupling reaction in absence of FA. After incubation for 2h, the beads were washed thoroughly with PBS to remove unbound proteins, SDS sample buffer was added and bead-bound proteins were released by heating at 95°C for 5 min. (B) For competition experiments target protein was mixed with competing protein in the concentrations as indicated in the figure before addition of beads. One representative blot out of 3 independent experiments is shown. Densities were evaluated using ImageJ. Results are given in mean±SD.
    Figure Legend Snippet: Pull down of FA-free HSA-0 and A1AT-0 by FA-coupled agarose beads. (A) Fatty acid free HSA-0 or A1AT-0 (Zemaira) was mixed on a rotating mixer with ω-aminohexyl-agarose beads coupled to LA, OA or control (co.) beads exposed to coupling reaction in absence of FA. After incubation for 2h, the beads were washed thoroughly with PBS to remove unbound proteins, SDS sample buffer was added and bead-bound proteins were released by heating at 95°C for 5 min. (B) For competition experiments target protein was mixed with competing protein in the concentrations as indicated in the figure before addition of beads. One representative blot out of 3 independent experiments is shown. Densities were evaluated using ImageJ. Results are given in mean±SD.

    Techniques Used: Incubation

    FA pull down of A1AT and HSA from PiMM and PiZZ serum. Pooled serum samples were pre-diluted with 3 volumes of PBS and incubated with LA-, OA- or control (co.) beads for 2 h on a rotating mixer. Beads were washed thoroughly with PBS to remove unbound proteins, SDS sample buffer was added and bead-bound proteins were released by heating at 95°C for 5 min. Sample load was 15 μl for A1AT blot and 5 μl for HSA blot. To facilitate comparability of the blots 200 ng target proteins were applied. Antibodies don't show any cross reactivity with competing proteins.
    Figure Legend Snippet: FA pull down of A1AT and HSA from PiMM and PiZZ serum. Pooled serum samples were pre-diluted with 3 volumes of PBS and incubated with LA-, OA- or control (co.) beads for 2 h on a rotating mixer. Beads were washed thoroughly with PBS to remove unbound proteins, SDS sample buffer was added and bead-bound proteins were released by heating at 95°C for 5 min. Sample load was 15 μl for A1AT blot and 5 μl for HSA blot. To facilitate comparability of the blots 200 ng target proteins were applied. Antibodies don't show any cross reactivity with competing proteins.

    Techniques Used: Incubation

    Qualitative analysis of molecular forms of A1AT found in A1AT preparations by non-denaturing SDS-PAGE following Western blot analysis using rabbit polyclonal anti-A1AT and mouse monoclonal anti-A1AT polymer (2C1) antibodies. (A) A1AT-LA and –OA complexes were prepared by 3 h incubation at 37°C following separation from free FA with centricon-10 (MWCO 10 kDa). Each blot is representative out of n=3 independent experiments. (B) M- and Z-A1AT pools from Alph1-Antitrypsin Select affinity purification were analyzed by Western Blot. Each blot is representative out of n=3 independent experiments.
    Figure Legend Snippet: Qualitative analysis of molecular forms of A1AT found in A1AT preparations by non-denaturing SDS-PAGE following Western blot analysis using rabbit polyclonal anti-A1AT and mouse monoclonal anti-A1AT polymer (2C1) antibodies. (A) A1AT-LA and –OA complexes were prepared by 3 h incubation at 37°C following separation from free FA with centricon-10 (MWCO 10 kDa). Each blot is representative out of n=3 independent experiments. (B) M- and Z-A1AT pools from Alph1-Antitrypsin Select affinity purification were analyzed by Western Blot. Each blot is representative out of n=3 independent experiments.

    Techniques Used: SDS Page, Western Blot, Incubation, Affinity Purification

    Time-dependent effect of A1AT-0 and A1AT-LA on Angptl4 and FABP4 expression of human adherent blood monocytes. Cells were treated with A1AT -0 or A1AT-LA (1 mg/ml) for different periods of time. Gene expression levels of Angptl4 (A) and FABP4 (B) were analyzed by RT-PCR and normalized to GAPDH. Each point represents mean ± SD of n=3 independent experiments, each with 3 repeats.
    Figure Legend Snippet: Time-dependent effect of A1AT-0 and A1AT-LA on Angptl4 and FABP4 expression of human adherent blood monocytes. Cells were treated with A1AT -0 or A1AT-LA (1 mg/ml) for different periods of time. Gene expression levels of Angptl4 (A) and FABP4 (B) were analyzed by RT-PCR and normalized to GAPDH. Each point represents mean ± SD of n=3 independent experiments, each with 3 repeats.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    Effects of A1AT-LA and A1AT-OA complexes on Angptl4 and related gene expression in adherent blood monocytes compared to those of A1AT-0 and FAs alone. Cells were treated with A1AT-0, complexes of A1AT-LA and A1AT-OA (1 mg/ml) or with LA and OA preparations alone for 6 h. Gene expression levels of Angptl4 (A), FABP4 (B) and CD36 (C) were assessed by RT-PCR and normalized to GAPDH. Each bar represents mean ± SD of n=6 independent experiments in case of A1AT-0, A1AT-LA and LA and n=3 in case of A1AT-OA and OA. Measurements were performed in triplicates.
    Figure Legend Snippet: Effects of A1AT-LA and A1AT-OA complexes on Angptl4 and related gene expression in adherent blood monocytes compared to those of A1AT-0 and FAs alone. Cells were treated with A1AT-0, complexes of A1AT-LA and A1AT-OA (1 mg/ml) or with LA and OA preparations alone for 6 h. Gene expression levels of Angptl4 (A), FABP4 (B) and CD36 (C) were assessed by RT-PCR and normalized to GAPDH. Each bar represents mean ± SD of n=6 independent experiments in case of A1AT-0, A1AT-LA and LA and n=3 in case of A1AT-OA and OA. Measurements were performed in triplicates.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    Effects of A1AT-LA and A1AT-OA complexes on Angptl4, FABP4 and CD36 protein levels. Adherent monocytes were kept untreated for the control or treated with A1AT-0, A1AT-LA or A1AT-OA complexes (1 mg/ml) for 6 h. (A) Supernatants were collected and analyzed for released Angptl4 by ARP4 ELISA. (B) Cells were fractionated into cytoplasmic and nuclear fractions using Cytoplasmic and Nuclear Fractionation kit. FABP4 protein levels in the fractions were determined by Western Blot analysis. β-Actin was stained for a loading control. Presented blot is representative of n=3 repeated experiments. Cell surface expression of CD36 was analyzed by flow cytometry (C). Cells were stained with anti CD36-FITC or respective FITC isotype. Mean fluorescence intensities of ungated cells were determined. Each bar represents mean ±SD of n=3 independent experiments, each performed in duplicates.
    Figure Legend Snippet: Effects of A1AT-LA and A1AT-OA complexes on Angptl4, FABP4 and CD36 protein levels. Adherent monocytes were kept untreated for the control or treated with A1AT-0, A1AT-LA or A1AT-OA complexes (1 mg/ml) for 6 h. (A) Supernatants were collected and analyzed for released Angptl4 by ARP4 ELISA. (B) Cells were fractionated into cytoplasmic and nuclear fractions using Cytoplasmic and Nuclear Fractionation kit. FABP4 protein levels in the fractions were determined by Western Blot analysis. β-Actin was stained for a loading control. Presented blot is representative of n=3 repeated experiments. Cell surface expression of CD36 was analyzed by flow cytometry (C). Cells were stained with anti CD36-FITC or respective FITC isotype. Mean fluorescence intensities of ungated cells were determined. Each bar represents mean ±SD of n=3 independent experiments, each performed in duplicates.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Fractionation, Western Blot, Staining, Expressing, Flow Cytometry, Fluorescence

    Effects of serum derived affinity purified M- and Z-A1AT on Angptl4 and related gene expression. Adherent blood monocytes were treated with 0.5 mg/ml of M- and Z-A1AT, isolated from pooled PiMM and PiZZ serum, for a total of 6 h. mRNA levels of Angptl4 (A), FABP4 (C) and CD36 (D) were analyzed by RT-PCR and normalized to GAPDH. Each point represents mean ±SD of n=4 independent experiments, each with 3 repeats. (B) Concentration of Angptl4 in cell culture supernatants was determined by Duoset ELISA. Each bar represents mean ± SD of n=4 independent experiments, each with 2 repeats.
    Figure Legend Snippet: Effects of serum derived affinity purified M- and Z-A1AT on Angptl4 and related gene expression. Adherent blood monocytes were treated with 0.5 mg/ml of M- and Z-A1AT, isolated from pooled PiMM and PiZZ serum, for a total of 6 h. mRNA levels of Angptl4 (A), FABP4 (C) and CD36 (D) were analyzed by RT-PCR and normalized to GAPDH. Each point represents mean ±SD of n=4 independent experiments, each with 3 repeats. (B) Concentration of Angptl4 in cell culture supernatants was determined by Duoset ELISA. Each bar represents mean ± SD of n=4 independent experiments, each with 2 repeats.

    Techniques Used: Derivative Assay, Affinity Purification, Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    A1AT-LA induced expression of Angptl4 and FABP4 is related to PPARγ and PPARβ/δ activity. Cells were pretreated for 30 min with 10 μM GW9662, an irreversible PPARγ antagonist (A, B), or with 1 μM ST247, a selective and inverse agonist of PPARβ/δ (C, D), prior to addition of 1 mg/ml A1AT-0, A1AT-LA, LA preparation or Prolastin® for another 6 h. Expression levels of Angptl4 (A, C) and FABP4 (B, D) were determined by RT-PCR and normalized to GAPDH. In (A, B) each point represents mean ±SD of n=3 independent experiments, each with 3 repeats. In (C, D) each point represents mean ±SD of n=2 independent experiments, each with 3 repeats.
    Figure Legend Snippet: A1AT-LA induced expression of Angptl4 and FABP4 is related to PPARγ and PPARβ/δ activity. Cells were pretreated for 30 min with 10 μM GW9662, an irreversible PPARγ antagonist (A, B), or with 1 μM ST247, a selective and inverse agonist of PPARβ/δ (C, D), prior to addition of 1 mg/ml A1AT-0, A1AT-LA, LA preparation or Prolastin® for another 6 h. Expression levels of Angptl4 (A, C) and FABP4 (B, D) were determined by RT-PCR and normalized to GAPDH. In (A, B) each point represents mean ±SD of n=3 independent experiments, each with 3 repeats. In (C, D) each point represents mean ±SD of n=2 independent experiments, each with 3 repeats.

    Techniques Used: Expressing, Activity Assay, Reverse Transcription Polymerase Chain Reaction

    (A) Transient activation of MEK-ERK1/2 pathway is independent on A1AT complexation with LA. Western blot of cell lysates prepared from adherent blood monocytes illustrates phosphorylation of ERK1/2 at 30 min in response to treatment with 1 mg/ml A1AT-0 or A1AT-LA. Blots were stained for phosphorylated and total ERK1/2. β-Actin was used as a loading control. Pre-incubation of cells with MEK/ERK1/2 inhibitor UO126 (10 μM), completely abolished ERK1/2 activation in all samples. Presented blot is representative of n=4 repeated experiments. (B) Effect of ERK1/2 phosphorylation on A1AT-LA-induced Angptl4 expression. Adherent PBMCs were pre incubated for 30 min with UO126 (10 μM) prior to addition of 1 mg/ml A1AT-0 or A1AT-LA for another 6 h. Angptl4 mRNA levels were assessed by RT-PCR and normalized to GAPDH. Each point represents mean ±SD of n=3 independent experiments, each with 3 repeats.
    Figure Legend Snippet: (A) Transient activation of MEK-ERK1/2 pathway is independent on A1AT complexation with LA. Western blot of cell lysates prepared from adherent blood monocytes illustrates phosphorylation of ERK1/2 at 30 min in response to treatment with 1 mg/ml A1AT-0 or A1AT-LA. Blots were stained for phosphorylated and total ERK1/2. β-Actin was used as a loading control. Pre-incubation of cells with MEK/ERK1/2 inhibitor UO126 (10 μM), completely abolished ERK1/2 activation in all samples. Presented blot is representative of n=4 repeated experiments. (B) Effect of ERK1/2 phosphorylation on A1AT-LA-induced Angptl4 expression. Adherent PBMCs were pre incubated for 30 min with UO126 (10 μM) prior to addition of 1 mg/ml A1AT-0 or A1AT-LA for another 6 h. Angptl4 mRNA levels were assessed by RT-PCR and normalized to GAPDH. Each point represents mean ±SD of n=3 independent experiments, each with 3 repeats.

    Techniques Used: Activation Assay, Western Blot, Staining, Incubation, Expressing, Reverse Transcription Polymerase Chain Reaction

    polymeric a1at  (Hycult Biotech)


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  • 86

    Structured Review

    Hycult Biotech polymeric a1at
    FA content in different <t> A1AT </t> preparations
    Polymeric A1at, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polymeric a1at/product/Hycult Biotech
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    polymeric a1at - by Bioz Stars, 2024-06
    86/100 stars

    Images

    1) Product Images from "Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression"

    Article Title: Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1500740

    FA content in different  A1AT  preparations
    Figure Legend Snippet: FA content in different A1AT preparations

    Techniques Used: Affinity Purification

    Pull down of FA-free HSA-0 and A1AT-0 by FA-coupled agarose beads. (A) Fatty acid free HSA-0 or A1AT-0 (Zemaira) was mixed on a rotating mixer with ω-aminohexyl-agarose beads coupled to LA, OA or control (co.) beads exposed to coupling reaction in absence of FA. After incubation for 2h, the beads were washed thoroughly with PBS to remove unbound proteins, SDS sample buffer was added and bead-bound proteins were released by heating at 95°C for 5 min. (B) For competition experiments target protein was mixed with competing protein in the concentrations as indicated in the figure before addition of beads. One representative blot out of 3 independent experiments is shown. Densities were evaluated using ImageJ. Results are given in mean±SD.
    Figure Legend Snippet: Pull down of FA-free HSA-0 and A1AT-0 by FA-coupled agarose beads. (A) Fatty acid free HSA-0 or A1AT-0 (Zemaira) was mixed on a rotating mixer with ω-aminohexyl-agarose beads coupled to LA, OA or control (co.) beads exposed to coupling reaction in absence of FA. After incubation for 2h, the beads were washed thoroughly with PBS to remove unbound proteins, SDS sample buffer was added and bead-bound proteins were released by heating at 95°C for 5 min. (B) For competition experiments target protein was mixed with competing protein in the concentrations as indicated in the figure before addition of beads. One representative blot out of 3 independent experiments is shown. Densities were evaluated using ImageJ. Results are given in mean±SD.

    Techniques Used: Incubation

    FA pull down of A1AT and HSA from PiMM and PiZZ serum. Pooled serum samples were pre-diluted with 3 volumes of PBS and incubated with LA-, OA- or control (co.) beads for 2 h on a rotating mixer. Beads were washed thoroughly with PBS to remove unbound proteins, SDS sample buffer was added and bead-bound proteins were released by heating at 95°C for 5 min. Sample load was 15 μl for A1AT blot and 5 μl for HSA blot. To facilitate comparability of the blots 200 ng target proteins were applied. Antibodies don't show any cross reactivity with competing proteins.
    Figure Legend Snippet: FA pull down of A1AT and HSA from PiMM and PiZZ serum. Pooled serum samples were pre-diluted with 3 volumes of PBS and incubated with LA-, OA- or control (co.) beads for 2 h on a rotating mixer. Beads were washed thoroughly with PBS to remove unbound proteins, SDS sample buffer was added and bead-bound proteins were released by heating at 95°C for 5 min. Sample load was 15 μl for A1AT blot and 5 μl for HSA blot. To facilitate comparability of the blots 200 ng target proteins were applied. Antibodies don't show any cross reactivity with competing proteins.

    Techniques Used: Incubation

    Qualitative analysis of molecular forms of A1AT found in A1AT preparations by non-denaturing SDS-PAGE following Western blot analysis using rabbit polyclonal anti-A1AT and mouse monoclonal anti-A1AT polymer (2C1) antibodies. (A) A1AT-LA and –OA complexes were prepared by 3 h incubation at 37°C following separation from free FA with centricon-10 (MWCO 10 kDa). Each blot is representative out of n=3 independent experiments. (B) M- and Z-A1AT pools from Alph1-Antitrypsin Select affinity purification were analyzed by Western Blot. Each blot is representative out of n=3 independent experiments.
    Figure Legend Snippet: Qualitative analysis of molecular forms of A1AT found in A1AT preparations by non-denaturing SDS-PAGE following Western blot analysis using rabbit polyclonal anti-A1AT and mouse monoclonal anti-A1AT polymer (2C1) antibodies. (A) A1AT-LA and –OA complexes were prepared by 3 h incubation at 37°C following separation from free FA with centricon-10 (MWCO 10 kDa). Each blot is representative out of n=3 independent experiments. (B) M- and Z-A1AT pools from Alph1-Antitrypsin Select affinity purification were analyzed by Western Blot. Each blot is representative out of n=3 independent experiments.

    Techniques Used: SDS Page, Western Blot, Incubation, Affinity Purification

    Time-dependent effect of A1AT-0 and A1AT-LA on Angptl4 and FABP4 expression of human adherent blood monocytes. Cells were treated with A1AT -0 or A1AT-LA (1 mg/ml) for different periods of time. Gene expression levels of Angptl4 (A) and FABP4 (B) were analyzed by RT-PCR and normalized to GAPDH. Each point represents mean ± SD of n=3 independent experiments, each with 3 repeats.
    Figure Legend Snippet: Time-dependent effect of A1AT-0 and A1AT-LA on Angptl4 and FABP4 expression of human adherent blood monocytes. Cells were treated with A1AT -0 or A1AT-LA (1 mg/ml) for different periods of time. Gene expression levels of Angptl4 (A) and FABP4 (B) were analyzed by RT-PCR and normalized to GAPDH. Each point represents mean ± SD of n=3 independent experiments, each with 3 repeats.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    Effects of A1AT-LA and A1AT-OA complexes on Angptl4 and related gene expression in adherent blood monocytes compared to those of A1AT-0 and FAs alone. Cells were treated with A1AT-0, complexes of A1AT-LA and A1AT-OA (1 mg/ml) or with LA and OA preparations alone for 6 h. Gene expression levels of Angptl4 (A), FABP4 (B) and CD36 (C) were assessed by RT-PCR and normalized to GAPDH. Each bar represents mean ± SD of n=6 independent experiments in case of A1AT-0, A1AT-LA and LA and n=3 in case of A1AT-OA and OA. Measurements were performed in triplicates.
    Figure Legend Snippet: Effects of A1AT-LA and A1AT-OA complexes on Angptl4 and related gene expression in adherent blood monocytes compared to those of A1AT-0 and FAs alone. Cells were treated with A1AT-0, complexes of A1AT-LA and A1AT-OA (1 mg/ml) or with LA and OA preparations alone for 6 h. Gene expression levels of Angptl4 (A), FABP4 (B) and CD36 (C) were assessed by RT-PCR and normalized to GAPDH. Each bar represents mean ± SD of n=6 independent experiments in case of A1AT-0, A1AT-LA and LA and n=3 in case of A1AT-OA and OA. Measurements were performed in triplicates.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    Effects of A1AT-LA and A1AT-OA complexes on Angptl4, FABP4 and CD36 protein levels. Adherent monocytes were kept untreated for the control or treated with A1AT-0, A1AT-LA or A1AT-OA complexes (1 mg/ml) for 6 h. (A) Supernatants were collected and analyzed for released Angptl4 by ARP4 ELISA. (B) Cells were fractionated into cytoplasmic and nuclear fractions using Cytoplasmic and Nuclear Fractionation kit. FABP4 protein levels in the fractions were determined by Western Blot analysis. β-Actin was stained for a loading control. Presented blot is representative of n=3 repeated experiments. Cell surface expression of CD36 was analyzed by flow cytometry (C). Cells were stained with anti CD36-FITC or respective FITC isotype. Mean fluorescence intensities of ungated cells were determined. Each bar represents mean ±SD of n=3 independent experiments, each performed in duplicates.
    Figure Legend Snippet: Effects of A1AT-LA and A1AT-OA complexes on Angptl4, FABP4 and CD36 protein levels. Adherent monocytes were kept untreated for the control or treated with A1AT-0, A1AT-LA or A1AT-OA complexes (1 mg/ml) for 6 h. (A) Supernatants were collected and analyzed for released Angptl4 by ARP4 ELISA. (B) Cells were fractionated into cytoplasmic and nuclear fractions using Cytoplasmic and Nuclear Fractionation kit. FABP4 protein levels in the fractions were determined by Western Blot analysis. β-Actin was stained for a loading control. Presented blot is representative of n=3 repeated experiments. Cell surface expression of CD36 was analyzed by flow cytometry (C). Cells were stained with anti CD36-FITC or respective FITC isotype. Mean fluorescence intensities of ungated cells were determined. Each bar represents mean ±SD of n=3 independent experiments, each performed in duplicates.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Fractionation, Western Blot, Staining, Expressing, Flow Cytometry, Fluorescence

    Effects of serum derived affinity purified M- and Z-A1AT on Angptl4 and related gene expression. Adherent blood monocytes were treated with 0.5 mg/ml of M- and Z-A1AT, isolated from pooled PiMM and PiZZ serum, for a total of 6 h. mRNA levels of Angptl4 (A), FABP4 (C) and CD36 (D) were analyzed by RT-PCR and normalized to GAPDH. Each point represents mean ±SD of n=4 independent experiments, each with 3 repeats. (B) Concentration of Angptl4 in cell culture supernatants was determined by Duoset ELISA. Each bar represents mean ± SD of n=4 independent experiments, each with 2 repeats.
    Figure Legend Snippet: Effects of serum derived affinity purified M- and Z-A1AT on Angptl4 and related gene expression. Adherent blood monocytes were treated with 0.5 mg/ml of M- and Z-A1AT, isolated from pooled PiMM and PiZZ serum, for a total of 6 h. mRNA levels of Angptl4 (A), FABP4 (C) and CD36 (D) were analyzed by RT-PCR and normalized to GAPDH. Each point represents mean ±SD of n=4 independent experiments, each with 3 repeats. (B) Concentration of Angptl4 in cell culture supernatants was determined by Duoset ELISA. Each bar represents mean ± SD of n=4 independent experiments, each with 2 repeats.

    Techniques Used: Derivative Assay, Affinity Purification, Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    A1AT-LA induced expression of Angptl4 and FABP4 is related to PPARγ and PPARβ/δ activity. Cells were pretreated for 30 min with 10 μM GW9662, an irreversible PPARγ antagonist (A, B), or with 1 μM ST247, a selective and inverse agonist of PPARβ/δ (C, D), prior to addition of 1 mg/ml A1AT-0, A1AT-LA, LA preparation or Prolastin® for another 6 h. Expression levels of Angptl4 (A, C) and FABP4 (B, D) were determined by RT-PCR and normalized to GAPDH. In (A, B) each point represents mean ±SD of n=3 independent experiments, each with 3 repeats. In (C, D) each point represents mean ±SD of n=2 independent experiments, each with 3 repeats.
    Figure Legend Snippet: A1AT-LA induced expression of Angptl4 and FABP4 is related to PPARγ and PPARβ/δ activity. Cells were pretreated for 30 min with 10 μM GW9662, an irreversible PPARγ antagonist (A, B), or with 1 μM ST247, a selective and inverse agonist of PPARβ/δ (C, D), prior to addition of 1 mg/ml A1AT-0, A1AT-LA, LA preparation or Prolastin® for another 6 h. Expression levels of Angptl4 (A, C) and FABP4 (B, D) were determined by RT-PCR and normalized to GAPDH. In (A, B) each point represents mean ±SD of n=3 independent experiments, each with 3 repeats. In (C, D) each point represents mean ±SD of n=2 independent experiments, each with 3 repeats.

    Techniques Used: Expressing, Activity Assay, Reverse Transcription Polymerase Chain Reaction

    (A) Transient activation of MEK-ERK1/2 pathway is independent on A1AT complexation with LA. Western blot of cell lysates prepared from adherent blood monocytes illustrates phosphorylation of ERK1/2 at 30 min in response to treatment with 1 mg/ml A1AT-0 or A1AT-LA. Blots were stained for phosphorylated and total ERK1/2. β-Actin was used as a loading control. Pre-incubation of cells with MEK/ERK1/2 inhibitor UO126 (10 μM), completely abolished ERK1/2 activation in all samples. Presented blot is representative of n=4 repeated experiments. (B) Effect of ERK1/2 phosphorylation on A1AT-LA-induced Angptl4 expression. Adherent PBMCs were pre incubated for 30 min with UO126 (10 μM) prior to addition of 1 mg/ml A1AT-0 or A1AT-LA for another 6 h. Angptl4 mRNA levels were assessed by RT-PCR and normalized to GAPDH. Each point represents mean ±SD of n=3 independent experiments, each with 3 repeats.
    Figure Legend Snippet: (A) Transient activation of MEK-ERK1/2 pathway is independent on A1AT complexation with LA. Western blot of cell lysates prepared from adherent blood monocytes illustrates phosphorylation of ERK1/2 at 30 min in response to treatment with 1 mg/ml A1AT-0 or A1AT-LA. Blots were stained for phosphorylated and total ERK1/2. β-Actin was used as a loading control. Pre-incubation of cells with MEK/ERK1/2 inhibitor UO126 (10 μM), completely abolished ERK1/2 activation in all samples. Presented blot is representative of n=4 repeated experiments. (B) Effect of ERK1/2 phosphorylation on A1AT-LA-induced Angptl4 expression. Adherent PBMCs were pre incubated for 30 min with UO126 (10 μM) prior to addition of 1 mg/ml A1AT-0 or A1AT-LA for another 6 h. Angptl4 mRNA levels were assessed by RT-PCR and normalized to GAPDH. Each point represents mean ±SD of n=3 independent experiments, each with 3 repeats.

    Techniques Used: Activation Assay, Western Blot, Staining, Incubation, Expressing, Reverse Transcription Polymerase Chain Reaction

    a1at polymeric forms  (Hycult Biotech)


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    Hycult Biotech a1at polymeric forms
    Summary of the nephelometric measurments of <t> A1AT </t> concentration in 966 samples analysed using routine serum protein electrophoresis (SPE) methods.
    A1at Polymeric Forms, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/a1at polymeric forms/product/Hycult Biotech
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    a1at polymeric forms - by Bioz Stars, 2024-06
    93/100 stars

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    1) Product Images from "How Can We Improve the Detection of Alpha1-Antitrypsin Deficiency?"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0135316

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

    Techniques Used: Concentration Assay, Protein Electrophoresis

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

    Techniques Used:

    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.
    Figure Legend 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.

    Techniques Used: Concentration Assay, Western Blot

    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.
    Figure Legend 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.

    Techniques Used: SDS Page, Western Blot, Concentration Assay

    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.
    Figure Legend 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.

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

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    Hycult Biotech a1at polymeric forms
    Summary of the nephelometric measurments of <t> A1AT </t> concentration in 966 samples analysed using routine serum protein electrophoresis (SPE) methods.
    A1at Polymeric Forms, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/a1at polymeric forms/product/Hycult Biotech
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    Hycult Biotech polymeric a1at
    FA content in different <t> A1AT </t> preparations
    Polymeric A1at, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Summary of the nephelometric measurments of  A1AT  concentration in 966 samples analysed using routine serum protein electrophoresis (SPE) methods.

    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

    Thresholds for suspicion of severe or intermediate A1ATD according to SPE.

    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:

    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.

    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

    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.

    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

    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.

    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

    FA content in different  A1AT  preparations

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression

    doi: 10.4049/jimmunol.1500740

    Figure Lengend Snippet: FA content in different A1AT preparations

    Article Snippet: For specific detection, the following primary antibodies were used: rabbit polyclonal anti A1AT (DAKO), mouse monoclonal anti A1AT (clone B9, Santa Cruz Biotechnology), mouse monoclonal anti polymeric A1AT (Clone 2C1, Hycult biotech), mouse monoclonal anti FABP4 (clone 1105CT1-1-1, Antibodies-Online GmbH), rabbit polyclonal anti ERK1/2 and mouse monoclonal anti p-ERK1/2 (both from Sigma-Aldrich), monoclonal anti β-actin (AC-15, Sigma-Aldrich).

    Techniques: Affinity Purification

    Pull down of FA-free HSA-0 and A1AT-0 by FA-coupled agarose beads. (A) Fatty acid free HSA-0 or A1AT-0 (Zemaira) was mixed on a rotating mixer with ω-aminohexyl-agarose beads coupled to LA, OA or control (co.) beads exposed to coupling reaction in absence of FA. After incubation for 2h, the beads were washed thoroughly with PBS to remove unbound proteins, SDS sample buffer was added and bead-bound proteins were released by heating at 95°C for 5 min. (B) For competition experiments target protein was mixed with competing protein in the concentrations as indicated in the figure before addition of beads. One representative blot out of 3 independent experiments is shown. Densities were evaluated using ImageJ. Results are given in mean±SD.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression

    doi: 10.4049/jimmunol.1500740

    Figure Lengend Snippet: Pull down of FA-free HSA-0 and A1AT-0 by FA-coupled agarose beads. (A) Fatty acid free HSA-0 or A1AT-0 (Zemaira) was mixed on a rotating mixer with ω-aminohexyl-agarose beads coupled to LA, OA or control (co.) beads exposed to coupling reaction in absence of FA. After incubation for 2h, the beads were washed thoroughly with PBS to remove unbound proteins, SDS sample buffer was added and bead-bound proteins were released by heating at 95°C for 5 min. (B) For competition experiments target protein was mixed with competing protein in the concentrations as indicated in the figure before addition of beads. One representative blot out of 3 independent experiments is shown. Densities were evaluated using ImageJ. Results are given in mean±SD.

    Article Snippet: For specific detection, the following primary antibodies were used: rabbit polyclonal anti A1AT (DAKO), mouse monoclonal anti A1AT (clone B9, Santa Cruz Biotechnology), mouse monoclonal anti polymeric A1AT (Clone 2C1, Hycult biotech), mouse monoclonal anti FABP4 (clone 1105CT1-1-1, Antibodies-Online GmbH), rabbit polyclonal anti ERK1/2 and mouse monoclonal anti p-ERK1/2 (both from Sigma-Aldrich), monoclonal anti β-actin (AC-15, Sigma-Aldrich).

    Techniques: Incubation

    FA pull down of A1AT and HSA from PiMM and PiZZ serum. Pooled serum samples were pre-diluted with 3 volumes of PBS and incubated with LA-, OA- or control (co.) beads for 2 h on a rotating mixer. Beads were washed thoroughly with PBS to remove unbound proteins, SDS sample buffer was added and bead-bound proteins were released by heating at 95°C for 5 min. Sample load was 15 μl for A1AT blot and 5 μl for HSA blot. To facilitate comparability of the blots 200 ng target proteins were applied. Antibodies don't show any cross reactivity with competing proteins.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression

    doi: 10.4049/jimmunol.1500740

    Figure Lengend Snippet: FA pull down of A1AT and HSA from PiMM and PiZZ serum. Pooled serum samples were pre-diluted with 3 volumes of PBS and incubated with LA-, OA- or control (co.) beads for 2 h on a rotating mixer. Beads were washed thoroughly with PBS to remove unbound proteins, SDS sample buffer was added and bead-bound proteins were released by heating at 95°C for 5 min. Sample load was 15 μl for A1AT blot and 5 μl for HSA blot. To facilitate comparability of the blots 200 ng target proteins were applied. Antibodies don't show any cross reactivity with competing proteins.

    Article Snippet: For specific detection, the following primary antibodies were used: rabbit polyclonal anti A1AT (DAKO), mouse monoclonal anti A1AT (clone B9, Santa Cruz Biotechnology), mouse monoclonal anti polymeric A1AT (Clone 2C1, Hycult biotech), mouse monoclonal anti FABP4 (clone 1105CT1-1-1, Antibodies-Online GmbH), rabbit polyclonal anti ERK1/2 and mouse monoclonal anti p-ERK1/2 (both from Sigma-Aldrich), monoclonal anti β-actin (AC-15, Sigma-Aldrich).

    Techniques: Incubation

    Qualitative analysis of molecular forms of A1AT found in A1AT preparations by non-denaturing SDS-PAGE following Western blot analysis using rabbit polyclonal anti-A1AT and mouse monoclonal anti-A1AT polymer (2C1) antibodies. (A) A1AT-LA and –OA complexes were prepared by 3 h incubation at 37°C following separation from free FA with centricon-10 (MWCO 10 kDa). Each blot is representative out of n=3 independent experiments. (B) M- and Z-A1AT pools from Alph1-Antitrypsin Select affinity purification were analyzed by Western Blot. Each blot is representative out of n=3 independent experiments.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression

    doi: 10.4049/jimmunol.1500740

    Figure Lengend Snippet: Qualitative analysis of molecular forms of A1AT found in A1AT preparations by non-denaturing SDS-PAGE following Western blot analysis using rabbit polyclonal anti-A1AT and mouse monoclonal anti-A1AT polymer (2C1) antibodies. (A) A1AT-LA and –OA complexes were prepared by 3 h incubation at 37°C following separation from free FA with centricon-10 (MWCO 10 kDa). Each blot is representative out of n=3 independent experiments. (B) M- and Z-A1AT pools from Alph1-Antitrypsin Select affinity purification were analyzed by Western Blot. Each blot is representative out of n=3 independent experiments.

    Article Snippet: For specific detection, the following primary antibodies were used: rabbit polyclonal anti A1AT (DAKO), mouse monoclonal anti A1AT (clone B9, Santa Cruz Biotechnology), mouse monoclonal anti polymeric A1AT (Clone 2C1, Hycult biotech), mouse monoclonal anti FABP4 (clone 1105CT1-1-1, Antibodies-Online GmbH), rabbit polyclonal anti ERK1/2 and mouse monoclonal anti p-ERK1/2 (both from Sigma-Aldrich), monoclonal anti β-actin (AC-15, Sigma-Aldrich).

    Techniques: SDS Page, Western Blot, Incubation, Affinity Purification

    Time-dependent effect of A1AT-0 and A1AT-LA on Angptl4 and FABP4 expression of human adherent blood monocytes. Cells were treated with A1AT -0 or A1AT-LA (1 mg/ml) for different periods of time. Gene expression levels of Angptl4 (A) and FABP4 (B) were analyzed by RT-PCR and normalized to GAPDH. Each point represents mean ± SD of n=3 independent experiments, each with 3 repeats.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression

    doi: 10.4049/jimmunol.1500740

    Figure Lengend Snippet: Time-dependent effect of A1AT-0 and A1AT-LA on Angptl4 and FABP4 expression of human adherent blood monocytes. Cells were treated with A1AT -0 or A1AT-LA (1 mg/ml) for different periods of time. Gene expression levels of Angptl4 (A) and FABP4 (B) were analyzed by RT-PCR and normalized to GAPDH. Each point represents mean ± SD of n=3 independent experiments, each with 3 repeats.

    Article Snippet: For specific detection, the following primary antibodies were used: rabbit polyclonal anti A1AT (DAKO), mouse monoclonal anti A1AT (clone B9, Santa Cruz Biotechnology), mouse monoclonal anti polymeric A1AT (Clone 2C1, Hycult biotech), mouse monoclonal anti FABP4 (clone 1105CT1-1-1, Antibodies-Online GmbH), rabbit polyclonal anti ERK1/2 and mouse monoclonal anti p-ERK1/2 (both from Sigma-Aldrich), monoclonal anti β-actin (AC-15, Sigma-Aldrich).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    Effects of A1AT-LA and A1AT-OA complexes on Angptl4 and related gene expression in adherent blood monocytes compared to those of A1AT-0 and FAs alone. Cells were treated with A1AT-0, complexes of A1AT-LA and A1AT-OA (1 mg/ml) or with LA and OA preparations alone for 6 h. Gene expression levels of Angptl4 (A), FABP4 (B) and CD36 (C) were assessed by RT-PCR and normalized to GAPDH. Each bar represents mean ± SD of n=6 independent experiments in case of A1AT-0, A1AT-LA and LA and n=3 in case of A1AT-OA and OA. Measurements were performed in triplicates.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression

    doi: 10.4049/jimmunol.1500740

    Figure Lengend Snippet: Effects of A1AT-LA and A1AT-OA complexes on Angptl4 and related gene expression in adherent blood monocytes compared to those of A1AT-0 and FAs alone. Cells were treated with A1AT-0, complexes of A1AT-LA and A1AT-OA (1 mg/ml) or with LA and OA preparations alone for 6 h. Gene expression levels of Angptl4 (A), FABP4 (B) and CD36 (C) were assessed by RT-PCR and normalized to GAPDH. Each bar represents mean ± SD of n=6 independent experiments in case of A1AT-0, A1AT-LA and LA and n=3 in case of A1AT-OA and OA. Measurements were performed in triplicates.

    Article Snippet: For specific detection, the following primary antibodies were used: rabbit polyclonal anti A1AT (DAKO), mouse monoclonal anti A1AT (clone B9, Santa Cruz Biotechnology), mouse monoclonal anti polymeric A1AT (Clone 2C1, Hycult biotech), mouse monoclonal anti FABP4 (clone 1105CT1-1-1, Antibodies-Online GmbH), rabbit polyclonal anti ERK1/2 and mouse monoclonal anti p-ERK1/2 (both from Sigma-Aldrich), monoclonal anti β-actin (AC-15, Sigma-Aldrich).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    Effects of A1AT-LA and A1AT-OA complexes on Angptl4, FABP4 and CD36 protein levels. Adherent monocytes were kept untreated for the control or treated with A1AT-0, A1AT-LA or A1AT-OA complexes (1 mg/ml) for 6 h. (A) Supernatants were collected and analyzed for released Angptl4 by ARP4 ELISA. (B) Cells were fractionated into cytoplasmic and nuclear fractions using Cytoplasmic and Nuclear Fractionation kit. FABP4 protein levels in the fractions were determined by Western Blot analysis. β-Actin was stained for a loading control. Presented blot is representative of n=3 repeated experiments. Cell surface expression of CD36 was analyzed by flow cytometry (C). Cells were stained with anti CD36-FITC or respective FITC isotype. Mean fluorescence intensities of ungated cells were determined. Each bar represents mean ±SD of n=3 independent experiments, each performed in duplicates.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression

    doi: 10.4049/jimmunol.1500740

    Figure Lengend Snippet: Effects of A1AT-LA and A1AT-OA complexes on Angptl4, FABP4 and CD36 protein levels. Adherent monocytes were kept untreated for the control or treated with A1AT-0, A1AT-LA or A1AT-OA complexes (1 mg/ml) for 6 h. (A) Supernatants were collected and analyzed for released Angptl4 by ARP4 ELISA. (B) Cells were fractionated into cytoplasmic and nuclear fractions using Cytoplasmic and Nuclear Fractionation kit. FABP4 protein levels in the fractions were determined by Western Blot analysis. β-Actin was stained for a loading control. Presented blot is representative of n=3 repeated experiments. Cell surface expression of CD36 was analyzed by flow cytometry (C). Cells were stained with anti CD36-FITC or respective FITC isotype. Mean fluorescence intensities of ungated cells were determined. Each bar represents mean ±SD of n=3 independent experiments, each performed in duplicates.

    Article Snippet: For specific detection, the following primary antibodies were used: rabbit polyclonal anti A1AT (DAKO), mouse monoclonal anti A1AT (clone B9, Santa Cruz Biotechnology), mouse monoclonal anti polymeric A1AT (Clone 2C1, Hycult biotech), mouse monoclonal anti FABP4 (clone 1105CT1-1-1, Antibodies-Online GmbH), rabbit polyclonal anti ERK1/2 and mouse monoclonal anti p-ERK1/2 (both from Sigma-Aldrich), monoclonal anti β-actin (AC-15, Sigma-Aldrich).

    Techniques: Enzyme-linked Immunosorbent Assay, Fractionation, Western Blot, Staining, Expressing, Flow Cytometry, Fluorescence

    Effects of serum derived affinity purified M- and Z-A1AT on Angptl4 and related gene expression. Adherent blood monocytes were treated with 0.5 mg/ml of M- and Z-A1AT, isolated from pooled PiMM and PiZZ serum, for a total of 6 h. mRNA levels of Angptl4 (A), FABP4 (C) and CD36 (D) were analyzed by RT-PCR and normalized to GAPDH. Each point represents mean ±SD of n=4 independent experiments, each with 3 repeats. (B) Concentration of Angptl4 in cell culture supernatants was determined by Duoset ELISA. Each bar represents mean ± SD of n=4 independent experiments, each with 2 repeats.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression

    doi: 10.4049/jimmunol.1500740

    Figure Lengend Snippet: Effects of serum derived affinity purified M- and Z-A1AT on Angptl4 and related gene expression. Adherent blood monocytes were treated with 0.5 mg/ml of M- and Z-A1AT, isolated from pooled PiMM and PiZZ serum, for a total of 6 h. mRNA levels of Angptl4 (A), FABP4 (C) and CD36 (D) were analyzed by RT-PCR and normalized to GAPDH. Each point represents mean ±SD of n=4 independent experiments, each with 3 repeats. (B) Concentration of Angptl4 in cell culture supernatants was determined by Duoset ELISA. Each bar represents mean ± SD of n=4 independent experiments, each with 2 repeats.

    Article Snippet: For specific detection, the following primary antibodies were used: rabbit polyclonal anti A1AT (DAKO), mouse monoclonal anti A1AT (clone B9, Santa Cruz Biotechnology), mouse monoclonal anti polymeric A1AT (Clone 2C1, Hycult biotech), mouse monoclonal anti FABP4 (clone 1105CT1-1-1, Antibodies-Online GmbH), rabbit polyclonal anti ERK1/2 and mouse monoclonal anti p-ERK1/2 (both from Sigma-Aldrich), monoclonal anti β-actin (AC-15, Sigma-Aldrich).

    Techniques: Derivative Assay, Affinity Purification, Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    A1AT-LA induced expression of Angptl4 and FABP4 is related to PPARγ and PPARβ/δ activity. Cells were pretreated for 30 min with 10 μM GW9662, an irreversible PPARγ antagonist (A, B), or with 1 μM ST247, a selective and inverse agonist of PPARβ/δ (C, D), prior to addition of 1 mg/ml A1AT-0, A1AT-LA, LA preparation or Prolastin® for another 6 h. Expression levels of Angptl4 (A, C) and FABP4 (B, D) were determined by RT-PCR and normalized to GAPDH. In (A, B) each point represents mean ±SD of n=3 independent experiments, each with 3 repeats. In (C, D) each point represents mean ±SD of n=2 independent experiments, each with 3 repeats.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression

    doi: 10.4049/jimmunol.1500740

    Figure Lengend Snippet: A1AT-LA induced expression of Angptl4 and FABP4 is related to PPARγ and PPARβ/δ activity. Cells were pretreated for 30 min with 10 μM GW9662, an irreversible PPARγ antagonist (A, B), or with 1 μM ST247, a selective and inverse agonist of PPARβ/δ (C, D), prior to addition of 1 mg/ml A1AT-0, A1AT-LA, LA preparation or Prolastin® for another 6 h. Expression levels of Angptl4 (A, C) and FABP4 (B, D) were determined by RT-PCR and normalized to GAPDH. In (A, B) each point represents mean ±SD of n=3 independent experiments, each with 3 repeats. In (C, D) each point represents mean ±SD of n=2 independent experiments, each with 3 repeats.

    Article Snippet: For specific detection, the following primary antibodies were used: rabbit polyclonal anti A1AT (DAKO), mouse monoclonal anti A1AT (clone B9, Santa Cruz Biotechnology), mouse monoclonal anti polymeric A1AT (Clone 2C1, Hycult biotech), mouse monoclonal anti FABP4 (clone 1105CT1-1-1, Antibodies-Online GmbH), rabbit polyclonal anti ERK1/2 and mouse monoclonal anti p-ERK1/2 (both from Sigma-Aldrich), monoclonal anti β-actin (AC-15, Sigma-Aldrich).

    Techniques: Expressing, Activity Assay, Reverse Transcription Polymerase Chain Reaction

    (A) Transient activation of MEK-ERK1/2 pathway is independent on A1AT complexation with LA. Western blot of cell lysates prepared from adherent blood monocytes illustrates phosphorylation of ERK1/2 at 30 min in response to treatment with 1 mg/ml A1AT-0 or A1AT-LA. Blots were stained for phosphorylated and total ERK1/2. β-Actin was used as a loading control. Pre-incubation of cells with MEK/ERK1/2 inhibitor UO126 (10 μM), completely abolished ERK1/2 activation in all samples. Presented blot is representative of n=4 repeated experiments. (B) Effect of ERK1/2 phosphorylation on A1AT-LA-induced Angptl4 expression. Adherent PBMCs were pre incubated for 30 min with UO126 (10 μM) prior to addition of 1 mg/ml A1AT-0 or A1AT-LA for another 6 h. Angptl4 mRNA levels were assessed by RT-PCR and normalized to GAPDH. Each point represents mean ±SD of n=3 independent experiments, each with 3 repeats.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression

    doi: 10.4049/jimmunol.1500740

    Figure Lengend Snippet: (A) Transient activation of MEK-ERK1/2 pathway is independent on A1AT complexation with LA. Western blot of cell lysates prepared from adherent blood monocytes illustrates phosphorylation of ERK1/2 at 30 min in response to treatment with 1 mg/ml A1AT-0 or A1AT-LA. Blots were stained for phosphorylated and total ERK1/2. β-Actin was used as a loading control. Pre-incubation of cells with MEK/ERK1/2 inhibitor UO126 (10 μM), completely abolished ERK1/2 activation in all samples. Presented blot is representative of n=4 repeated experiments. (B) Effect of ERK1/2 phosphorylation on A1AT-LA-induced Angptl4 expression. Adherent PBMCs were pre incubated for 30 min with UO126 (10 μM) prior to addition of 1 mg/ml A1AT-0 or A1AT-LA for another 6 h. Angptl4 mRNA levels were assessed by RT-PCR and normalized to GAPDH. Each point represents mean ±SD of n=3 independent experiments, each with 3 repeats.

    Article Snippet: For specific detection, the following primary antibodies were used: rabbit polyclonal anti A1AT (DAKO), mouse monoclonal anti A1AT (clone B9, Santa Cruz Biotechnology), mouse monoclonal anti polymeric A1AT (Clone 2C1, Hycult biotech), mouse monoclonal anti FABP4 (clone 1105CT1-1-1, Antibodies-Online GmbH), rabbit polyclonal anti ERK1/2 and mouse monoclonal anti p-ERK1/2 (both from Sigma-Aldrich), monoclonal anti β-actin (AC-15, Sigma-Aldrich).

    Techniques: Activation Assay, Western Blot, Staining, Incubation, Expressing, Reverse Transcription Polymerase Chain Reaction