Recombinant Anti-beta 2 Microglobulin antibody [EP2978Y] (ab75853)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.	-Mouse (1:1000) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Upstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.
Protocol tips
-Mouse (1:1000) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

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 Abcam for Recombinant Anti-beta 2 Microglobulin antibody [EP2978Y] (ab75853) below.

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