1. After I lyse the mammalian cells, do I have to do the assay right away? May I freeze the samples in -80C freezer and do the assays later?

Answer: You may freeze the samples at -80C after they're lysed, and do the assays altogether.

2. Why PBS is used to dilute the Standard? Since the samples are in the lysis buffer, do you think I should use the lysis buffer to dilute them?

Answer: PBS is commonly used. People usually use the lysis buffer to lyse the cells, then use PBS to dilute the samples. The point is that you should keep the same concentration of the lysis buffer for the samples and the Standards.

3. I did a comparison, and it showed that the PBS based Standard curve looked better than the lysis buffer based Standard curve. What do you think of this?

Answer: The bubbles in the lysis buffer may cause the inconsistency of the readings.

4. When I do duplicates of Standards, the reading varies a lot in the low range (0.2 uM or lower). Why does that happen?

Answer: It is normal. There are many factors that would cause inconsistent readings, i.e. bubbles in the wells, the volumes.... So try doing more replicates.

5. Sometimes for the 2 sets of Standards I set up, they look good (R2=0.99) individually but the curve is messed up (R2=0.95) when merged. So which curve should I use to quantitate my data?

Answer: You'll need to use the curve set up on the same plate with your samples.

6. We don't have fluorescence microplate reader, so can we use absorbance microplate reader?

Answer: Yes. But the absorbance readings have much lower sensitivity compared to the fluorescence readings. Try using the ratio of 570 to 610 to increase the sensitivity.

7. We do not have 540 nm excitation filter but we have 560 nm. We don't have the absorbance 573 nm. What is the range we could possible use?

Answer: We recommend to use 560/590, and cutoff 570 nm; OR 560/610, cutoff 590 nm.