TIMP2 (D18B7) Rabbit mAb #5738

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:1000)

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.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Western blotting TIMP-2 using TIMP2 (D18B7) Rabbit mAb #5738 from Cell Signaling Technology.

Manufacturer protocol

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

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Western blotting TIMP-2 using TIMP2 (D18B7) Rabbit mAb #5738 from Cell Signaling Technology. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.