I had run a phos-tag electrophoresis with resolving gel contain 6% of acrylamide, 50 uM of phos-tag, 100 µM ZnCl2, TEMED, 0.35M of Bis-Tris base and APS. I ran the gel at 15mA/gel for 4hours. But what I got is the phosphorylated protein does not well separated (please refer to the figureas attached). My target protein size is 100 kDa. Do I need to run the gel at lower mA for overnight or increase the concentration of Phos-Tag?
Hope you guys can give me some suggestion. =)
The molecular mass of your protein is indeed rather high to be detected by binding to a small phos-tag, especially if you expect the presence of the single phosphorylation site in your protein. Note also, that this method provides worser resolution for for detection the phosphorylatied tyrosine residue.
Nevertheless, please, try running at 5 mA about 12-18 h to get the difference. For better resolution 4% gel might be better.
Other details regarding the application of the method to large proteins you can find at this site: https://www.nature.com/protocolexchange/protocols/501#/related-articles
By reducing the bisacrilamide in your gel prep you may get a better seperation it worth trying. Please look for recepies of low cross linking acrylamide gels
Hi,
I would suggest starting with adjusting the concentration of phos-tag. Some Phos-tag tool kits recommend testing 20, 50, 100 and 150 uM phos-tag to establish the best conditions for separation of your target proteins. Increasing Phos-tag concetration might require using higher % of resolving gel though.
Good luck!
Best,
Joanna
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