goat polyclonal antibodies  (R&D Systems)

Journal: eLife

Article Title: Dynamics of compartment-specific proteomic landscapes of hepatotoxic and cholestatic models of liver fibrosis

doi: 10.7554/eLife.98023

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were used: rat mAb to K19 (Troma III; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA); mouse mAbs to αSMA (DAKO, Glostrup, Denmark) and ECM1 (Santa Cruz Biotechnology, Dallas, TX, USA), goat polyclonal antibodies to clusterin (R&D Systems, Minneapolis, MN, USA), rabbit polyclonal antibodies to fibronectin, collagen type I, collagen type IV, and monoclonal to integrin αV (all from Abcam, Cambridge, UK).

Techniques: Software, Staining

Journal: Neuroscience letters

Article Title: Methionine oxidation of clusterin in Alzheimer's disease and its effect on clusterin's binding to beta-amyloid.

doi: 10.1016/j.neulet.2024.137874

Figure Lengend Snippet: Fig. 3. MetO-Clusterin levels are predominantly expressed in postmortem AD-model mouse brains as a function of age. A. Postmortem mouse brain samples at 4 months of age (Lanes 1 & 3) and at 8 months of age; Lanes 2 & 4). Lanes 3 & 4 represent protein loads of 5xFAD mouse model of AD and Lanes 1 & 2 represent protein loads of the corresponding controls. Whole brains (n = 5 per strains per age) were extracted in PBS in the presence of protease inhibitors (Sigma-Aldrich). The resulting extracted protein moiety contains both cellular and extracellular proteins in each brain. Equal amounts of protein extracts per brain were subjected to immunoprecipitation (IP), using the rabbit anti-MetO antibody; followed by western-blot (WB) analysis, using a primary goat anti-mouse clusterin antibody (Pro teintech). The WB depicts representative data for one brain per strain per age. kDa, molecular mass markers. B. Western blot analysis of whole brain protein extracts obtained for the brains described in panel A (using the same goat anti-mouse clusterin primary antibody). (Lanes 1 & 3) and at 8 months of age; Lanes 2 & 4). Lanes 3 & 4 represent protein loads of 5xFAD mouse model of AD and Lanes 1 & 2 represent protein loads of the corresponding controls. The WB depicts representative data for one brain per strain per age (total n = 5 per strainper age). The β-actin expression levels serve as loading controls (using anti-β-actin primary antibody, Thermo- Fisher Scientific). kDa, molecular mass markers. C. Quantification of the clusterin and MetO-Clusterin bands shown in the WB in Fig. 3 A&B (using the NIH-Image-J program). The data showed that the 5xFAD brains had higher MetO-Clusterin levels relative to the non-AD brains (*, t-test, P<0.001) after correction for the total expressed levels of clusterin in panel B.

Article Snippet: Equal amounts of protein extracts per brain were subjected to immunoprecipitation (IP), using the rabbit anti-MetO antibody [1]; followed by WB analysis (Fig. 3A), using a primary goat anti-mouse clusterin antibody (Proteintech, Rosemont, IL).

Techniques: Immunoprecipitation, Western Blot, Expressing

Journal: Neuroscience letters

Article Title: Methionine oxidation of clusterin in Alzheimer's disease and its effect on clusterin's binding to beta-amyloid.

doi: 10.1016/j.neulet.2024.137874

Figure Lengend Snippet: Fig. 2. MetO-Clusterin levels are predominantly expressed in postmortem brains of late-stage AD patients. A. Postmortem human brain samples of late stages of AD (hippocampus region, n = 5) and at late stages of non-AD brains (hippocampus region, n = 5) were incubated in PBS in the presence of 0.32 M sucrose and protease inhibitors (Sigma-Aldrich) for 4 h at 4 ◦C. Equal amounts of the resulting extracellular protein moiety from each brain were subjected to immunoprecipitation (IP), using the rabbit anti-MetO antibody; followed by western-blot (WB) analysis, using a primary mouse anti-human clusterin antibody (Proteintech). Lanes 1–3, loads of one processed extracted brain per each lane (representatives out of the total brains’ number). B. Western blot analysis of extracellular brain protein extracts obtained for the brains described in panel A (using the same mouse anti-human clusterin antibody). Equal amounts of protein extracts were loaded per each lane. C. Quantification of the clusterin and MetO-Clusterin bands shown in the WB in Fig. 2 (using the NIH-Image-J program) showed that the AD brains had about 3.5-fold higher MetO-Clusterin levels relative to the non-AD brains (*, t-test, P<0.001) after correction for the total expressed levels of clusterin in panel B.

Article Snippet: Equal amounts of protein extracts per brain were subjected to immunoprecipitation (IP), using the rabbit anti-MetO antibody [1]; followed by WB analysis (Fig. 3A), using a primary goat anti-mouse clusterin antibody (Proteintech, Rosemont, IL).

Techniques: Incubation, Immunoprecipitation, Western Blot

Journal: Neuroscience letters

Article Title: Methionine oxidation of clusterin in Alzheimer's disease and its effect on clusterin's binding to beta-amyloid.

doi: 10.1016/j.neulet.2024.137874

Figure Lengend Snippet: Fig. 4. Oxidation of active recombinant ApoJ and the binding kinetics of re combinant ApoJ (clusterin) and its oxidized form (MetO-ApoJ) to Aβ42, in the presence of ThT. A. Methionine oxidation of an active ApoJ protein (50 µg) was performed by incubating the protein with 50 mM H2O2 for 3 h at room tem perature. The residual H2O2 was quenched by multiple washes in PBS and concentration using an Amicon Ultra-0.5 Centrifugal Filter Unit (3000 Da cut- off). The oxidation of the methionine moiety of ApoJ was confirmed by both dot-blot (right panel) and western blot (left panel) analyses using the anti-MetO antibody (as the primary antibody) and 1µg of non-oxidized and oxidized ApoJ. N, non-oxidized ApoJ; Ox, oxidized ApoJ; kDa, molecular mass markers in kilo- Daltons. B. Effects of ApoJ and MetO-ApoJ on the kinetics of Aβ42 fibril for mation. Freshly prepared Aβ42 was diluted at the indicated concentration in 20 mM phosphate buffer (pH 8.0) containing 0.02 % NaN3 and 200 µM EDTA. Then the Aβ42 was incubated, in the presence and absence of active recombi nant human ApoJ or MetO-ApoJ, under quiescent conditions at 37 ◦C in microplate 96-wells Microplate. in the presence of 20 µM ThT. ThT fluorescence was measured every 4.0 min for 18 h, using an M200 Infinity plate reader. A parallel analysis was performed using BSA instead ApoJ, at the indicated con centration, serving as a non-chaperone control protein. The ThT dye was excited at 440 nm, and the emission was measured at 495 nm. Fluorescence data are reported after normalization on the corresponding maximal ThT value after subtracting ThT basal background signal value. The figure shows a representative data of three repetitive independent experiments. Abeta, Aβ42.

Article Snippet: Equal amounts of protein extracts per brain were subjected to immunoprecipitation (IP), using the rabbit anti-MetO antibody [1]; followed by WB analysis (Fig. 3A), using a primary goat anti-mouse clusterin antibody (Proteintech, Rosemont, IL).

Techniques: Recombinant, Binding Assay, Concentration Assay, Dot Blot, Western Blot, Incubation, Fluorescence, Control

Journal: Neuroscience letters

Article Title: Methionine oxidation of clusterin in Alzheimer's disease and its effect on clusterin's binding to beta-amyloid.

doi: 10.1016/j.neulet.2024.137874

Figure Lengend Snippet: Fig. 5. In-Silico analyses for the location of clusterin’s conserved methionine residues and their associated hydrophobicity regions. The number indicates the position of a methionine residue in the sequence alignment. The intensity of the red color indicates the level of hydrophobicity, and the intensity of the blue color indicates the level of hydrophilicity (a denser color-coded amino acids corelates with either stronger hydrophobic or hydrophilic cluster, respectively). The Uniprot program and the align tool were used to acquire the clusterin sequences of the indicated organisms. The amino acid numbers are based on the longest Clusterin sequence in the indicated species.

Article Snippet: Equal amounts of protein extracts per brain were subjected to immunoprecipitation (IP), using the rabbit anti-MetO antibody [1]; followed by WB analysis (Fig. 3A), using a primary goat anti-mouse clusterin antibody (Proteintech, Rosemont, IL).

Techniques: In Silico, Residue, Sequencing