I do ELISA many times, and my result is not the same. I realize that when I harvested the supernatant, the volume each well was different. For example I stimulate cells with 200 ul media, when I harvest it, the volume in each well is different and less than 200 ul. So my concern is, should I make all the supernatant at the same volume (200 ul) by adding media, then dilute it with blocking buffer as I want? Or just dilute with blocking buffer directly without making the same volume first?
Thank you very much 🙏
In enzyme-linked immunosorbent assay (ELISA) procedures, it's important to maintain consistency in sample volumes to ensure accurate and reliable results. If you lose some volume during the harvest of supernatant, it's generally advisable to top up or adjust the volumes to make them uniform before dilution.
Here are a few reasons why maintaining consistent volumes is crucial in ELISA:
Before topping up, it's essential to consider the potential impact on the concentration of the analyte. If topping up significantly dilutes the sample, it could affect the assay's sensitivity. Ideally, any adjustments to volumes should be made while keeping the overall dilution factor in mind.
Always follow the specific guidelines provided by the ELISA kit manufacturer or your laboratory protocol for sample handling, as they may have specific recommendations based on the characteristics of the assay.
- Intan Mp If there are variations in the volumes collected, you may consider add the wells with additional media to make the volumes consistent across all samples. Adjust the volumes to a uniform level (e.g., 200 µL) for each well.
Intan Mp It would be better to prevent a strong unequal evaporation rather than simply adjusting volumes afterwards since this may irreversibly affect your data.
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