i bought solid alpha-glucosidase enzyme with the following details on its label.
750UN
alpha-glucosidase from Saccharomyces cerevisiae
recombinant expressed in proprietary host, lyophilized powder
>100 units/mg protein
15.3 mg solid
94 units/mg protein (maltose)
23% protein (buiret)
215 units/mg protein
how do i dissolved the solid to get the final concentration of 0.025 unit/well?
thank you in advance for your reply
What volume of the enzyme solution do you intend to put in each well?
Will the concentration of the phosphate buffer affect the result? Because i am using 30mM of phosphate buffer to run my essay. What is the best pH for alpha-glucosidase? I am preparing my phosphate buffer with a pH of 6.5
0.025 U/well in 10 µl/well is 0.0025 U/µl or 2.5 U/ml. This is the final concentration of enzyme in the well. Assuming you have to make at least one other addition to the well, namely the substrate, you may add the enzyme at 2X the final concentration, hence 5 U/ml. You probably don't want to make a single stock solution of 750 U at 5 U/ml, which is 150 ml. So, if you want to dissolve all of the solid at once, you should make a concentrated stock solution, as suggested by Jorg. If you make a 200X solution (500 U/ml), you would dissolve all the enzyme in 1.5 ml of an appropriate buffer, divide it into a number of small aliquots, flash freeze them, and store them at -80C.
Amended on 01 April 2017
Lysosomal alpha-glucosidase (human) has hydrophobicity of 0.589, which contains 7 N-glycoside chains. alpha-Glucosidase from Saccharomyces cerevisiae has hydrophobicity of 0.492, but may have only one N-glycoside chain.
Human milk biotinidase (with O-glycochains) and porcine cerebrum biotinidase (with N-glycoside chains; please see file; brain BIN pig) have hydrophobicities of 0.505 and 0.504, respectively, and non-ionic surfactant (NP-40) is necessary in order to dissolve these biotinidases fully (please see file; IEF for hydrophobic protein).
By the way, human insulin-like growth factor-1 (IGF-1) has hydrophobicity of 0.600. Human insulin has hydrophobicity of 0.607. Somatotropin (Growth hormone; human) has no-glycochains, and has hydrophobicity of 0.487. Growth hormone-binding protein (GHBP; human) has 5 N-glycochains and has hydrophobicity of 0.482. Then, these non-glycosylated proteins except GHBP should also be dissolved in the non-ionic surfactant containing solvent.
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