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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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separation of phospho and dephosphoprotein
Posted by: Yunsik Choi (IP Hidden, New member, 3)
Date: September 6, 2005 08:04AM
Hi. I'm trying to separate phospho and dephospho proteins from mouse hippocampus using one antibody. This antibody can detect both forms and there is no specific antibody available. The results were always one band. I run so long time to increase separation capacity. My first question is whether sonication is bad choice for western of phosphoprotein. Another question is about the condition. Protein size is about 46 kD. So can you let me know which gel percent and what voltage will be good.
Please help me. Thanks.
Re: separation of phospho and dephosphoprotein
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: September 6, 2005 10:09AM
1. Use phosphatase inhibitors in your lysis buffer. In some cases the phosphorylated proteins can be dephosphorlated quickly. Sigma and other vendors sale inhibitor cocktails for tyrosine and serine/threonine phosphotases.
2. The separation of two forms depends on phosphorylation condition of your protein. More phosphorylation sites will lead to more mobility shift. 3. You can run 2D IEF gel followed by western blot to confirm the phosphorylation. It's an expensive alternative and is more convincing. If you know the upstream kinase and you have recombinant protein, you can also try the in vitro kinase assay and load both cold and hot protein in your gel. Sonication shouldn't be the problem. Normal lysis condition should work fine though. My two cents.
Re: separation of phospho and dephosphoprotein
Posted by: Yunsik Choi (IP Hidden, New member, 3)
Date: September 6, 2005 01:19PM
Thank you very much.
But can I ask which is better between fast running with higher voltage or slow running with lower voltage? Sincerely. Edited 1 times. Last edit at 09/06/05 01:28PM by Yunsik Choi.
Re: separation of phospho and dephosphoprotein
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: September 7, 2005 08:34AM
Yes protein runs faster at higer voltage and slower at lower voltage. It won't help you to enhance the separation resolution. Other types of acrylamide gel may help to increase the resolution: gradient gel, Tricine gel etc.
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