HDL fraction separated by SDS-polyacrylamide gel electrophoresis was transferred to polyvinylidene fluoride membrane. After blocking with 5 % milk, the membrane was incubated overnight at 4 °C with anti-apoA-I (Santa cruz biotechnology) oranti-Nε-(carboxyethyl)-lysine (CEL) antibody (Cosmo Bio Co., Tokyo, Japan). Then, ECL reagent (GE Healthcare, UK) was used for detection. Films were examined using an HP Scanjet Pro flatbed scanner, and images were analyzed and quantified with Adobe Photoshop CS2 software. Absolute intensity was assessed via multiplying the mean density value by pixel for each band, and relative intensity of apoA-I glycation was calculated as absolute intensity of apoA-I glycation divided by that of apoA-I protein.
Anti apoa 1
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-ApoA-1 is a laboratory reagent used for the detection and quantification of Apolipoprotein A-I (ApoA-1) in biological samples. ApoA-1 is a major component of high-density lipoprotein (HDL) cholesterol and plays a key role in lipid metabolism. The Anti-ApoA-1 product can be used in various research and diagnostic applications to measure ApoA-1 levels.
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Lab products found in correlation
Anti vitronectin, by Abcam (2 mentions)
Ecl reagent, by GE Healthcare (1 mentions)
Anti n myc, by Santa Cruz Biotechnology (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
Anti c myc, by Abcam (1 mentions)
Anti synaptophysin, by Santa Cruz Biotechnology (1 mentions)
Anti synapsin 1, by Abcam (1 mentions)
Anti syntaxin 1b, by Synaptic Systems (1 mentions)
Anti apoe, by Abcam (1 mentions)
Protein g sepharose 4 fast flow beads, by GE Healthcare (1 mentions)
Anti tsg101, by Santa Cruz Biotechnology (1 mentions)
Na934v, by GE Healthcare (1 mentions)
Na931v, by GE Healthcare (1 mentions)
Anti alix, by Cell Signaling Technology (1 mentions)
Anti flot 1, by BD (1 mentions)
Anti cd9, by Cell Signaling Technology (1 mentions)
Anti rabbit igg hrp, by Thermo Fisher Scientific (1 mentions)
Anti rat igg hrp, by Thermo Fisher Scientific (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Anti alix, by Santa Cruz Biotechnology (1 mentions)
8 protocols using anti apoa 1
1
ApoA-I Glycation Quantification by SDS-PAGE
2
Western Blot Analysis of Protein Expression
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Cell lysates were prepared with RIPA buffer supplemented with protease and phosphatase inhibitors. The protein concentration was determined with the Bradford assay. Equal amounts of protein (20 μg) were loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. The protein was then transferred to polyvinylidene difluoride (PVDF) membranes followed by blocking with 5% nonfat milk. The following primary antibodies were applied with overnight incubation: anti-ApoA-I (1:200 dilution; Santa Cruz), anti-N-Myc (1:200 dilution; Santa Cruz), anti-c-Myc (1:1000 dilution; Abcam, Cambridge, MA, USA), and anti-actin (1:200 dilution; Santa Cruz).
3
Reverse Co-Immunoprecipitation of Synaptic Proteins
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For reverse co-IPs, protein G-Sepharose 4 Fast Flow beads (GE Healthcare) were pre-bound with 4 μg of the following antibodies, respectively: anti-mitochondrial creatine kinase U-type (MtCK, Abcam, San Francisco, catalog no. ab76506); anti-β-synuclein (Abcam, catalog no. ab76111); anti-apoE (Abcam, catalog no. ab1906); anti-apoAI (Santa Cruz Biotechnology, Dallas, TX, catalog no. sc-30089); anti-syntaxin 1B (Synaptic Systems, Göttingen, Germany, catalog no. 110402); anti-synapsin 1 (Abcam, catalog no. ab8); anti-synaptogyrin-3 (Santa Cruz Biotechnology, catalog no. sc-68936); anti-synaptophysin (Santa Cruz Biotechnology, catalog no. sc-9116); and anti-synaptotagmin (Millipore, catalog no. MAB5200). These samples were then incubated with 1 mg of lysate at 4 °C overnight. To verify the binding preference of Tau isoforms, dephosphorylated lysates were used adapting a dephosphorylation protocol obtained from New England Biolabs. Beads were washed extensively with IP washing buffer and eluted in 2× SDS-PAGE sample buffer for subsequent Western blot analysis. Blots were probed with the following antibodies: anti-creatine kinase U-type; mitochondrial (MtCK); anti-β-synuclein; anti-apoE; anti-apoAI; anti-syntaxin 1B; anti-synapsin 1; anti-synaptogyrin-3; anti-synaptophysin; anti-synaptotagmin, and anti-Tau. For quantification, ImageJ was used.
4
Extracellular Vesicle Protein Immunostaining
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The following antibodies were used for immunostaining: anti-Alix (1:1000, 2171 S, Cell Signaling Technology, Beverly, Massachusetts, USA), anti-TSG101 (1:1000, sc-7964, Santa Cruz Biotechnology, Dallas, Texas, USA), anti-CD9 (1:1000, D3H4P, Cell Signaling Technology, Beverly, Massachusetts, USA), anti-THP (1:800, sc-20631, Santa Cruz Biotechnology, Dallas, Texas, USA), anti-Flot-1 (1:1000, 610820, BD Biosciences, Franklin Lakes, New Jersey, USA), anti-Ago2 (1:1000, ab32381, Abcam, Cambridge, UK), anti-ApoA-1 (1:100, B10, Santa Cruz Biotechnology, Dallas, Texas, USA), sheep anti-mouse horseradish peroxidase-linked antibody (1:3000, NA931V, GE Healthcare Life Sciences, Uppsala, Sweden), donkey anti-rabbit horseradish peroxidase-linked antibody (1:4000, NA934V, GE Healthcare Life Sciences, Uppsala, Sweden).
5
Extracellular Vesicle Protein Analysis
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For western blot analysis, an equal volume of the EV fractions were mixed with Laemmli buffer (Bio-Rad Laboratories) and boiled at 95°C for 5 minutes. Samples were then loaded on a 10% SDS-PAGE gel and transferred onto PVDF membrane (Bio-Rad Laboratories). The membranes were blocked with 5% non-fat milk dissolved in PBS for 1 h and then probed with primary antibodies overnight at 4°C (primary antibodies: anti-CD9 (1:100, Abcam), anti-Flotillin (1:500, Cell Signaling), anti-Alix (1:100, Santa Cruz Biotechnology), anti-Calnexin (1:500, Abcam), anti-GM130 (1:250, BD Biosciences), anti-CD81 (1:100, Santa Cruz Biotechnology), anti-CD63 (1:100, BD Pharmingen), anti-APOA1 (1:500, Santa Cruz Biotechnology). After 4 washes in PBS containing 0.1% Tween (PBST), membranes were incubated with corresponding HRP-conjugated secondary antibodies: anti-mouse IgG-HRP (1:1000, Santa Cruz Biotechnology), anti-rabbit IgG-HRP (1:1000, Thermo Fisher Scientific) or anti-rat IgG-HRP (1:1000, Thermo Fisher Scientific) for 1h and washed in PBST. Signals were visualized after incubation with Amersham ECL Prime substrate and imaged using an ImageQuant LAS 4000.
6
Quantitative Western Blot Analysis
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Cells were lysed and total protein was estimated by using DC™ Protein Assay Kit (Bio‐Rad). Mice plasma was diluted with PBS in a ratio of 1:10 before gel separation. Proteins were resolved in 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membrane (Millipore). Membranes were incubated in 5% nonfat dry milk, followed by incubation with a primary and secondary antibody with three alternate wash. Blots were finally incubated with NBT and BCIP in alkaline phosphatase buffer until the bands were visible. The primary antibodies used for the study were anti‐vitronectin (1:1,000) (cat no. 45139; Abcam), anti‐transferrin (1:10,000) (cat no. Ab82411; Abcam), anti‐β‐actin (1:10,000) (cat. no. A5441; Sigma), anti‐ApoA2 (1:1000) (cat. no. A14690; Ab clonal), anti‐ApoA1 (1:1000) (cat no. sc‐376818; Santa Cruz), IL‐1β (1:1000) (cat no. sc‐52012; Santa Cruz), MCP‐1 (1:2000) (cat no. A7277; Ab clonal). Quantitative analysis was done by Image J software. The ratio of levels of experimental protein/loading control was used for the evaluation of relative levels of the protein.
7
Quantitative ELISA for Biomarker Detection
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We followed indirect and quantitative ELISA method.18 (link),19 (link) Plasma samples from RA, OA and HC were coated into 96-well microtiter plates (Nunc, USA), incubated with anti-TTR, anti-Serotransferrin, anti-ApoA1 and anti-RAGE antibodies (Santa Cruz, USA) separately followed by HRP-conjugated secondary antibody (Jackson, USA) incubation. For quantitative ELISA, commercialized available ELISA kit for TTR (Elabscience, E-EL-H2342), RAGE (Ray-Bio, ELH-RAGE) and ACPAs (EH4137) were used according to manufactures guideline (see details in supplementary methods).
8
Immunoblot Analysis of Apolipoproteins
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20 µg of non-depleted pooled samples were loaded into pre-cast tris-gly gels AnyKD (Bio-Rad Laboratories Inc © , Hercules, CA, USA). After electrophoresis, the gels were incubated in transfer buffer (Bio-Rad Laboratories Inc © , Hercules, CA, USA) for 10 min. Transfer was performed in a Bio-Rad minitransfer system using a PVDF membrane previously activated with methanol for 1.5 h at 200 mA. After transfer, the gels were fixed in 40% ethanol / 10% acetic acid and stained. Membranes were blocked overnight with 5 % BSA (Roche Diagnostics © , S.L, Basilea, Switzerland) in TBS buffer (Bio-Rad Laboratories Inc © , Hercules, CA, USA) at 4º C. Membrane washing was performed with TBS buffer-Tween20 (5%). The primary antibodies were monoclonal antibodies anti-Apo A1 (Santa Cruz Biotechnology, Inc © , Dallas, TX, USA), antikininogen 1 (Abcam © , Cambridge, UK) and anti-vitronectin (Abcam © , Cambridge, UK) all diluted in 5% TBS-Tween20.
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