my sampel(plant ethanolic extract)did not dissolve in 10% dmso.. i try 40% but still unable to dissolve.. i also use sonicator, but still unable to dissolve..
1.) first solution
dilute using pure dmso
2.) second solution
dilute my sampel using ethanol..
do pure dmso or ethanol leave any effect to alpha glucosidase assay?
According to the protocol, I have incubated the mixture of 10 ul of sample diluted in pure dmso+10ul 10%dmso+enzyme (0.5 M) + 50ul phosphate buffer for 10 min. Then, I added 25 ul of PNPG and incubated the solution for another 30 min. To terminate the reaction, I have added 100 ul of 0.2 M of sodium carbonate and the absorbance was read at 410 nm.
is there any problem with this procedure?
In general, any organic solvent has a strong possibility of impacting any enzymatic assay. The NIH, for instance, generally recommends using no more than 2-2.5% DMSO final concentration in enzymatic assays. So while you can certainly dilute your substance in pure DMSO, you'd want to use only a small bit of it in a water-based reaction. So your reaction sounds a bit high in DMSO concentration to me.
And, of course, you can test whether the solvent effects your assay easily enough: simply run the assay at 0, 2, 5, 10, 25, 50, 100% DMSO or Ethanol, for instance, and see how the readout changes. When the readout starts changing by 5-10% or more relative to the 0% organic control, you've probably hit the absolute upper limit for how much of that organic solvent you should allow to incubate with your protein.
Dr. William P Katt is right.
You can use MeOH to dissolve your samples, then dilute by buffer.
In my case, 50% MeOH in buffer was used as a negative control.
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