Ab52945, by Abcam - Product details

1

Immunostaining and Histological Analysis of Transplant Tissue

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Formalin-fixed tissue was paraffinized, embedded, and cut into five-micron sections. Sections were stained with hematoxylin and eosin and assessed by an experienced transplant pathologist using the Suzuki score (Table 3) [37 (link)].
Immunofluorescence was used to confirm the results of the global proteomics analysis. We studied the expression of apolipoprotein A1 (ApoA1) and platelet–endothelial cell adhesion molecule (PECAM)-1 according to the Opal protocol (PerkinElmer/Akoya, Waltham, MA, USA) according to manufacturers’ instructions using primary antibodies against ApoA1 (#ab52945, Abcam, UK) and PECAM-1 (#ab281583, Abcam). Slides were then examined blindly by an experienced pathologist, and protein overall expression was evaluated semiquantitatively from weak (+) to strong (+++). The results were then correlated with the proteomics (Spearman).

Norén Å., Oltean M., Friman S., Molinaro A., Mölne J., Sihlbom C., Herlenius G, & Thorsell A. (2022). Liver Graft Proteomics Reveals Potential Incipient Mechanisms behind Early Renal Dysfunction after Liver Transplantation. International Journal of Molecular Sciences, 23(19), 11929.

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2

Quantification of SAA and apoA-I Proteins

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For Figure 1A, the SAA protein (3 µg) of the lipid-free state was loaded and electrophoresed on 15% SDS-PAGE gels and detected using the anti-SAA antibody (SC20651, SC biotech, Santa Cruz, CA, USA) as the primary antibody (diluted 1:2000) and goat anti-rabbit immunoglobulin G-horseradish peroxidase (HRP) (A120-101P, Bethyl Laboratories, Montgomery, TX, USA) as the secondary antibody (diluted 1:5000). For Figure 6B, glycated apoA-I and multimerized apoA-I were detected using the anti-apoA-I antibody (Ab52945, Abcam, London, UK) as the primary antibody. The protein concentrations in the lipid-free and lipid-bound states were determined using the Bradford assay modified by Markwell et al. [52 (link)] with bovine serum albumin as a standard. Band intensities (BI) were compared by band scanning with Chemi-Doc^® XR (Bio-Rad, Hercules, CA, USA) using Quantity One software (version 4.5.2). The BI was compared using Student’s t-test on the SPSS program from three independent blots.

, & Cho K.H. (2022). Human Serum Amyloid a Impaired Structural Stability of High-Density Lipoproteins (HDL) and Apolipoprotein (Apo) A-I and Exacerbated Glycation Susceptibility of ApoA-I and HDL. Molecules, 27(13), 4255.

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3

AAV-Mediated Overexpression of ApoA1 in Cell Lines

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Rat BOEC, RAEC, RASMC and Raw264.7 cells were transduced with AAV2-apoA1(WT) and AAV2-apoA1(4WF) at MOI of 10⁵–5 × 10⁵ (10¹⁰–3.5 × 10¹⁰ VG/ml). The respective culture media were formulated using a lipoprotein-depleted FBS. The media were collected after 3 days of culture, and the concentration of human apoA1 in the conditioned media was assayed with ELISA. For immunofluorescence studies, RAEC and RASMC were transduced with AAV2-apoA1(WT) and AAV2-apoA1(4WF) as above. Three days post-transduction the cells were fixed in cold methanol and were stained with anti-apoA1 antibody (Abcam, ab52945), followed by either Alexa-488 or Alexa-549 labelled secondary antibodies. Properly stained untransduced RAEC and apoA1-transduced RASMC not exposed to the primary antibody served as controls.

Hooshdaran B., Pressly B.B., Alferiev I.S., Smith J.D., Zoltick P.W., Tschabrunn C.M., Wilensky R.L., Gorman R.C., Levy R.J, & Fishbein I. (2022). Stent-based delivery of AAV2 vectors encoding oxidation-resistant apoA1. Scientific Reports, 12, 5464.

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4

Exosomal Protein Identification by Western Blot

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Exosomal protein (20 μg) was separated by SDS-PAGE, transferred onto a nitrocellulose membrane, probed with each primary antibody, and incubated with horseradish peroxidase (HRP)-linked secondary antibody. Blots were visualized with enhanced chemiluminescence (ECL) detection reagents and quantified using ECL hyperfilm. Band volumes were measured by densitometry in at least three different experiments. Primary antibodies were used against the following proteins: tetraspanin-1 (TSPAN1) (H00010103, 1:500; Abnova), hemopexin (HPX) (ab124935, 1:500; Abcam), polymeric immunoglobulin receptor (PIGR) (ab91269, 1:500; Abcam), apolipoprotein A-I (APOA1) (ab52945, 1:500; Abcam), and lectin galactoside-binding soluble 3 binding protein (LGALS3BP) (ab123921, 1:500; Abcam).

Lim J.H., Lee C.H., Kim K.Y., Jung H.Y., Choi J.Y., Cho J.H., Park S.H., Kim Y.L., Baek M.C., Park J.B., Kim Y.H., Chung B.H., Lee S.H, & Kim C.D. (2018). Novel urinary exosomal biomarkers of acute T cell-mediated rejection in kidney transplant recipients: A cross-sectional study. PLoS ONE, 13(9), e0204204.

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5

Immunoblotting Analysis of HDL Proteins

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Classical SDS-PAGE migration (12% polyacrylamide) was carried out in order to separate the proteins contained in the HDL fraction, followed by a liquid transfer to nitrocellulose membrane, as previously described^{52 (link)}. The following primary antibodies were used: anti-paraoxonase 1 (Abcam, ab24261, used at 1:1000 dilution = 2 μg/mL), anti-serum amyloid A-1 (Abcam, ab190802, used at 1:10,000 dilution = 42 ng/mL), anti-alpha-1 antitrypsin (Calbiochem, catalog no. 178260, used at 1:1,000), anti-ApoA-I (Abcam, ab52945 used at 1:4,000 = 47.5 ng/mL).

Begue F., Tanaka S., Mouktadi Z., Rondeau P., Veeren B., Diotel N., Tran-Dinh A., Robert T., Vélia E., Mavingui P., Lagrange-Xélot M., Montravers P., Couret D, & Meilhac O. (2021). Altered high-density lipoprotein composition and functions during severe COVID-19. Scientific Reports, 11, 2291.

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6

Western Blot Analysis of APOA1

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Native gels that were not silver stained were submitted for Western blot analysis. The gels were transferred using iBlot dry transfer (Thermo Fisher Scientific, Waltham, MA) at 25 V for 6 min. The membranes were then incubated in water at 4 °C until ready to block. Blocking was done using a solution of 5% goat serum (MP biomedicals, Hyderabad, India) in 0.5% Tween 20 tris-buffered saline (TBS-T) for 1 h at room temperature. Rabbit anti-APOA1 (ab52945, Abcam, Cambridge, UK) was used as the primary antibody and was added to the 5% goat serum in TBS-T solution at 1/5000; the membrane was left to incubate with the primary antibody overnight at 4 °C. Following primary incubation, the membranes were washed with TBS-T 3 times for 5 min each. The membranes were then blocked with a secondary goat anti-rabbit antibody at 1/10000 for 2 h at room temperature. Following secondary incubation, the membranes were again washed with TBS-T 3 times for 5 min. Luminata Crescendo HRP (Merk, Burlington, Massachusetts) substrate was then used to incubate the membrane for ~2 min, and the membrane was then analyzed using an EZ Gel Imager (BioRad, Hercules, CA). The membrane was analyzed with the Chemi HiResolution protocol, with images taken every 10 s for 10 contiguous minutes.

Lloyd-Jones C., Seckler H.D., DiStefano N., Sniderman A., Compton P.D., Kelleher N.L, & Wilkins J.T. (2023). Preparative Electrophoresis for HDL Particle Size Separation and Intact-Mass Apolipoprotein Proteoform Analysis. Journal of proteome research, 22(5), 1455-1465.

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7

Western Blot Analysis of APOA1

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Native gels that were not silver stained were submitted for Western blot analysis. The gels were transferred using iBlot dry transfer (Thermo Fisher Scientific, Waltham, MA) at 25 V for 6 min. The membranes were then incubated in water at 4 °C until ready to block. Blocking was done using a solution of 5% goat serum (MP biomedicals, Hyderabad, India) in 0.5% Tween 20 tris-buffered saline (TBS-T) for 1 h at room temperature. Rabbit anti-APOA1 (ab52945, Abcam, Cambridge, UK) was used as the primary antibody and was added to the 5% goat serum in TBS-T solution at 1/5000; the membrane was left to incubate with the primary antibody overnight at 4 °C. Following primary incubation, the membranes were washed with TBS-T 3 times for 5 min each. The membranes were then blocked with a secondary goat anti-rabbit antibody at 1/10000 for 2 h at room temperature. Following secondary incubation, the membranes were again washed with TBS-T 3 times for 5 min. Luminata Crescendo HRP (Merk, Burlington, Massachusetts) substrate was then used to incubate the membrane for ~2 min, and the membrane was then analyzed using an EZ Gel Imager (BioRad, Hercules, CA). The membrane was analyzed with the Chemi HiResolution protocol, with images taken every 10 s for 10 contiguous minutes.

Lloyd-Jones C., Dos Santos Seckler H., DiStefano N., Sniderman A., Compton P.D., Kelleher N.L, & Wilkins J.T. (2023). Preparative Electrophoresis for HDL Particle Size Separation and Intact-Mass Apolipoprotein Proteoform Analysis. Journal of proteome research, 22(5).

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Ab52945

Lab products found in correlation

7 protocols using ab52945

Immunostaining and Histological Analysis of Transplant Tissue

Quantification of SAA and apoA-I Proteins

AAV-Mediated Overexpression of ApoA1 in Cell Lines

Exosomal Protein Identification by Western Blot

Immunoblotting Analysis of HDL Proteins

Western Blot Analysis of APOA1

Western Blot Analysis of APOA1

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