I want to make colorimetric quantification of Lipase enzyme using Lowry's reagent. But while preparing the sample solutions (Lipase enzyme concentration of 1mg/ml) of different concentration of Lipase in distilled water I found that it is partially soluble in distilled water and some precipitate remains at the bottom.
Any suggestions?
It is soluble in water and in dilute buffer, according to the literature. I would try adding 10mg of Lipase to 10mls of deionised water or Phosphate buffered saline, pH 7.4. shaking and ultrasonication of this will aid dissolution.
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I hope this helps.
You could dissolve it in a detergent solution. The Lowry assay is compatible with most detergents. Or you could make a stock solution at a lower concentration at which lipase is soluble. Or you could make the stock solution in a buffer in which it is more soluble than in distilled water.
Even if there is still some undissolved protein, if you can make a homogeneous suspension, then you could use it in the assay if it subsequently dissolves in the Lowry A reagent.
Lipase will not dissolve in water, it is in contact with solvent ( water or THF or hexan) to perform reactions according to my knowledge.
The above are fine. The difficulty is that lipases can easily form aggregates in aqueous buffers. You could try dispersing your protein solution (in n-butanol or detergent solution) through a fine needle into the assay buffer. Just a suggestion
I find that the dissolution problems are usually caused by the enzyme or protein 'clumping' or aggregating when the water or buffer is added to dry powder (making it almost impossible to dissolve satisfactorily). I try to add the enzyme/protein crystals/powder to about 50-75% of the final volume of the buffer, in a beaker, with stirring - it may take a while to dissolve, so a magnetic stirrer might be preferable - adding most of the remains of the buffer volume, via the weighing receptacle, to ensure all of the powder is transferred to the beaker and dissolved. I would then pour it into a volumetric flask, washing it out of the beaker with the last of the buffer, to the final volume.
I hope this helps with the lipase solubilisation.
Dear SD,
You can use PBS or Tris buffer to make the solution of your lipase. It can be quantified by Lowry assay.
Best luck
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