I have a problem of precipitate formation after adding KI/iodine solution on starch solution, preventing absorbance measurement, anyone can kindly help to solve this problem?
Are you are talking about Somogyi assay? Starch, adding enzyme solution, incubation, KI/I2 solution, dilution with water and photometric measuring at visible wavelenghts 550 or 610 nm. If concentration of starch and iodine is to high it can precipitate out of solution. Therefore I think that you are using too high concentration of reactant(s) or possibly a wrong reactant. What kind of starch are you using?
I'd personally recommend a different assay, particularly if you need kinetic data rather than an endpoint assay. Two suggestions are below:
1. Purchase a defined maltose polymer (ex. maltohexaose). They are more soluble than starch. Carry out coupled assays with alpha-glucosidase, glucose oxidase and horseradish peroxidase. It gives you a nice color change in a real-time format.
2. Purchase a defined maltose polymer with a para-nitrophenol fused to the end. Carry out a coupled assay with alpha-glucosidase. This substrate is more expensive than the one in #1.
Thanks Srdjan for your answer. Starch is soluble starch. The reaction is Amylase + extract + .5% starch 15 min incubation. Addition of KI-Iodine solution (0.083g KI + 0.1269 g Iodine + 100ml water). Reading at 595 nm but I am not sure this is a good wave length..any suggestions?
Precipitate is first problem you have to solve. If precipitation happen in negative probe that does not contain amylase then concentration you are using are way to high and you should dilute your sample until there is no visible precipitation. As a rule of thumb best region for absorption measurement is between 0.3 and 0.7. Find a concentration range so that your negative probe described above have A=0.7. Find a wavelength scan of starch - iodine complex from literature. Record a wavelength scan of your sample with visible precipitate. Usually sample with precipitate have wide absoprtion due to scattering of light on particles and that is your indication during dilution does precipitation is still happening. Closer you get to literature wavelength scan the better. I can only think of one other thing that could be reason for precipitate. I assume that you are using stopping of enzyme reaction with boiling. If protein concentration is to high maybe they are responsible for precipitate? If that is the case then you could either dilute that to or just centrifuge your sample and measure absorption of supernatant. You are adding iodine solution to cooled reaction mixture I guess?
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