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
Western blot
Immunostaining
Not Rated
IP
Not Rated
ELISA
Not Rated
Flow cytometry
Not Rated
Luminex
Not Rated
On October 6, 2015Mads Kjolby wrote:
4
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.
On December 4, 2017 Simon Mølgaard wrote:
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
On October 6, 2015 Mads Kjolby wrote:
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.