May I know 400U of lambda phosphatase can dephosphorylate how many microgram of protein? I am using lambda phosphatase from Cosmo Bio.
Hi there,
It will depend on protein MW (1µg of protein is not the same if the protein is 10kDa or 100kDa... You will have 10 times less copies for the latter) and the actual level of protein phosphorylation. As a reference you can consider the NEB PPase which is obviously very similarly conditioned compared to the CosmoBio one:
100 units of Lambda PP remove ~100% of phosphates (0.5 nmol) in phosphorylated myelin basic protein (phospho-MyBP, 18.5 kDa) in 30 minutes in a 50 µl reaction. The concentration of phospho-MyBP is 10 µM with respect to phosphate.
For more info:
https://www.neb.com/products/p0753-lambda-protein-phosphatase-lambda-pp#pd-properties-usage
Will the incubation time affect the result? My protein is ~100kDa and I used 20ug of protein with addition of 200U of phosphatase. Incubation time of 1 hour is sufficient?
Hi again,
Of course incubation length will affect the result as long as the desphosphorylation is not completed. 20µg of a 100kDa protein is 0.2 nmol. According to NEB's leaflet your are within the condition used in the control experiment with phospho-MyBP (0.5nmol of phosphorylated ser/thr) if your protein contains one unique or two phosphorylated sites. The problem is actually how many phosphorylation sites have you on your protein? If only less than 3, you can start with condition described for phospho-MyBP. The main issue will be accessibility of the phosphorylated sites to the enzyme (may be you will have to consider extending incubation time or using more PPase if phospho sites are less reactive with the enzyme). If more phosphorylated sites, the condition you described might be tested (more enzyme and longer incubation). Well you can't validate the condition before having actually performed any assay! So analysis of your first attempt will tell you what to do...
Thank you very much for your information! I got the results that I wanted finally.
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