TIMP2 (D18B7) Rabbit mAb #5738

Western blotting TIMP-2

Experiment
Western blotting TIMP-2
Product
TIMP2 (D18B7) Rabbit mAb #5738 from Cell Signaling Technology
Manufacturer
Cell Signaling Technology

Protocol tips

Protocol tips
-Mouse (1:1000)

Publication protocol

Pulldown of Gpr158 was performed in solubilized membranes from Ocn−/− or Gpr158−/− hippocampi. Hippocampi were dissected on ice and homogenized in buffer A (10 mM Tris-HCl, pH 7.4, 320 mM sucrose, 1× protease, and phosphatase inhibitor cocktail [78443; Thermo Fisher Scientific]) with a Glass/Teflon Potter Elvehjem homogenizer (20 strokes). Homogenized hippocampi were centrifuged at 3,000 g for 10 min at 4°C. Then, supernatants were ultracentrifuged at 40,000 g for 20 min at 4°C. Pellets were resuspended in buffer A supplemented with 150 mM NaCl and 1% n-Octyl β-D-thioglucopyranoside (28310; Thermo Fisher Scientific). Solubilized membranes were diluted in buffer A supplemented with 150 mM NaCl and 0.2% n-Octyl β-D-thioglucopyranoside. Recombinant uncarboxylated OCN was biotynalated using the EZ-Link NHS-PEO4 biotynalation kit (21455; Thermo Fisher Scientific) according to the manufacturer’s instructions. For the pulldown, biotinylated OCN (7 μg) was incubated for 2 h at 4°C. Thirty microliters of Dynabeads M-280 Streptavidin (11205D; Thermo Fisher Scientific) were added for 30 min at room temperature followed by three PBS 1× washes. Purified proteins were eluted from the beads by adding Laemmli protein buffer and heated at 65°C for 15 min and analyzed by Western blot.

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for TIMP2 (D18B7) Rabbit mAb #5738 below.

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