Department of Biophysics
Panjab University
As part of my work, I was in a search of a user-friendly and sensitive protocol for the evaluation of sialic acid as I am working on phytochemicals in the area of cancer research. I wanted to analyze the effect of a combined treatment of phytochemicals on sialic acid levels. Over-expression of sialic acid leads to the generation of an excess negative charge on the cell membrane surface which in turn aids metastasis and the progression of cancer. I tested and evaluated various protocols. I rated them with respect to sensitivity and specificity as well as other aspects, such as ease-of-use, hands-on time, the level of complexity of results interpretation and the required skill level of the user.
The chemistry utilized in the Sialic Acid Assay Kit from Abcam involves an enzyme-coupled reaction in which free sialic acid is oxidized. This results in the development of the oxired probe that fluoresces (Ex/Em= 535/587 nm) and can also be read via absorbance (OD=570 nm). The kit measures sialic acid in the linear range of 0.1 - 10 nmol with a detection sensitivity of 1 µM concentration.
I found the instructions to be clear and concise so that anyone skilled in basic laboratory technique could perform the assay. Secondly, using the same kit, it is possible to work with both fluorescent and visible absorption. Also, the protocol is sensitive enough to allow estimation in range of micromoles levels.
My research involves use of tissue homogenates. Prior to dissection, lung tissues were perfused with Tris Buffer, pH 7.4 to remove any red blood contamination. Tissue homogenates were made followed by centrifugation at 10.000 rpm for 15 minutes at 4°C. The resultant supernatant was used for the above assay. Then, as directed in the kit, different reagents, such as sialic acid assay buffer, sialic acid probe in DMSO, sialic acid converting enzyme and sialic acid development mix solutions were prepared. To perform the assay, I first added 2 µl of sample to each well of 96-well plate and then made the volume to 50 µl with assay buffer. I then added 2 µl each of sialic acid converting enzme, sialic acid development mix and sialic acid probe. I incubated the reaction mixture at room temperature for 30 min with constant shaking. This was followed by measuring at an OD of 570 nm using a visible spectrophotometer.
The results I obtained were highly reproducible as we often repeat a particular experiment twice and take the readings in triplets. So, as to check the reproducibility, we ran experiments with this kit three times and found readings to be very stable with minimal variation in the readings, The statistical significance of the results were of level of 0.001 analyzed by statics software SPSS 14, using ANOVA statistic test. The kit also ranked good in terms of sensitivity and specificity as it can allow estimation of the samples in the micromole range.
I recommend the Sialic acid Assay Kit.