Phospho-GSK-3β, Cell Signaling Technology, 9322

This antibody was tested on 50 ug lysate from mouse primary hippocampal neurons.
A clear and intense signal was observed at the correct size after a very short exposure time. I used a concentration of 1:1000. The buffer used to lyse the cells is shown below:

• 20 mM MOPS, pH 7.0 (any other buffer at this pH could be substituted);
• 2 mM EGTA (to bind calcium);
• 5 mM EDTA (to bind magnesium and manganese);
• 30 mM sodium fluoride (to inhibit protein-serine phosphatases);
• 60 mM β-glycerophosphate, pH 7.2 (to inhibit protein-serine phosphatases);
• 20 mM sodium pyrophosphate (to inhibit protein-serine phosphatases);
• 1 mM sodium orthovanadate (to inhibit protein-tyrosine phosphatases);
• 1% Triton X-100 (can be substituted with 1% Nonidet P-40) Important Note: Do not add if you intend to first prepare a cytosolic fraction.
• 1 mM phenylmethylsulfonylfluoride (to inhibit proteases);
• 3 mM benzamidine (to inhibit proteases);
• 5 µM pepstatin A (to inhibit proteases);
• 10 µM leupeptin (to inhibit proteases);
• 1 mM dithiothreitol (to reduce disulphide linkages)

adjust to pH 7.2

I have used anti-phosphoGK3beta Ser9 (CST #9322s) at 1:1000 (milk based buffer) on primary murine macrophages. I get a nice band at 45 kDa as expected.