This antibody was tested on 50 ug lysate from mouse primary hippocampal neurons.
The antibody was tested simultaneously as p-JNK from SantaCruz #6254 (see full review here: http://pabmabs.com/wordpress/?p=866). While #6254 did not work at all, an excellent result was obtained using this antibody. 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)
On November 30, 2017 Simon Mølgaard wrote:
This antibody was tested on 50 ug lysate from mouse primary hippocampal neurons.
The antibody was tested simultaneously as p-JNK from SantaCruz #6254 (see full review here: http://pabmabs.com/wordpress/?p=866). While #6254 did not work at all, an excellent result was obtained using this antibody. 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