Gel-purified platelets cells were stimulated with 160 μM AYPGKF for 5 min, 15 min, 30 min, 1 h at 37 °C and 0 time (vehicle control). Samples were pretreated platelets with 30 μM AZD1283, +160 μM AYPGKF for 5 min, 15 min, 30 min, 1 h at 37 °C and 0 time (vehicle control). Prepared platelet lysates using extraction lysate buffer (25 mM tris(hydroxymethyl)aminomethane (Tris)-HCl at pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1% Nonidet P-40, 1 mM DTT (dithiothreitol), 5% Glycerol, 1 g/mL aprotinin, 1 g/mL leupeptin, and 1 mM phenyl methylsulphonyl fluoride (PMSF)) on ice for 10 min, cells were centrifuged at 14,000 g for 10 min at 4 °C. Aliquots of the lysate were used to detect total Rap1 levels. The remaining supernatants were incubated with 50 μL of Ral GDS-RBD agarose slurry of (EMD Millipore, MA) for 60 min at 4 °C. Proteins were separated on a 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to nitrocellulose membranes (BioRad, Hercules, CA). Rap1 levels were detected with rabbit polyclonal antibodies (EMD Millipore, MA) followed by horseradish peroxidase (HRP)–conjugated goat anti-rabbit antibodies (Zymed, South San Francisco, CA).
Rabbit polyclonal antibodies
Manufactured by Merck Group
Rabbit polyclonal antibodies are a type of laboratory reagent produced by immunizing rabbits with specific antigens. These antibodies recognize and bind to multiple epitopes on the target antigen, providing a broad spectrum of reactivity. Polyclonal antibodies are commonly used in various immunoassay techniques, such as ELISA, Western blotting, and immunohistochemistry, to detect and quantify the presence of target proteins in biological samples.
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Lab products found in correlation
2 protocols using rabbit polyclonal antibodies
1
Rap1 Activation in Stimulated Platelets
2
Immunostaining for Kidney Injury Markers
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An indirect immunoperoxidase method was used to identify the antigens, as previously described16 (link). Macrophage infiltration was identified using rat monoclonal antibody F4/80 (1:200, BMA Biomedicals, Augst, Switzerland). Type I and III collagens were identified using rabbit polyclonal antibodies (Cedarlane Laboratories, Burlington, ON, Canada). To identify cell proliferation, rat monoclonal antibody against proliferating cell nuclear antigen (PCNA; DAKO, Carpinteria, CA, USA) was used. Aquaporin-1, as a proximal tubular marker, was identified with the use of rabbit polyclonal antibodies (Merck, Darmstadt, German). Five to ten nonoverlapping fields from the cortical and outer medullary areas in each object were examined under × 100 magnification for F4/80, Type I and III collagens, and Aquaporin-1, and under × 200 magnification for PCNA. Positively stained areas for these antibodies were measured with WinRoof and expressed as ratios to the total examined areas. Brightness and contrast were applied equally across the entire image with image editor GIMP 2.10.14. (https://www.gimp.org ). Data were shown as the fold-increase or -decrease in positively stained areas in IR-kidneys compared with sham kidneys on day 3, 14, and 28 postischemia or with non-IR kidneys on day 70 postischemia.
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