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polymeric a1at (Hycult Biotech)

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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Average 86 stars, based on 1 article reviews
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polymeric a1at - by Bioz Stars, 2023-12

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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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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