Hi everyone,
I have a kit to analyse if our compounds inhibit an epigenetic reader protein, BRD4.
Kit is from Cayman (Cat No 600520). The kit says “It is recommended that inhibitor compounds to be tested in a concentration response format with at least eight independent concentrations that…”
It is the first time I have heard independent concentration?does it have any special meaning? I always prepare serial dilutions. But independent means different method?
Thank you
If you do serial dilution, the error of measurement multiplies with each step. Hence, further dilutions will become more and more imprecise, which you probably won't take into consideration when determining the average means of your results. Though serial dilution being rather convenient. If you require highly precise measurements (requiring equally precise detection methods), don't do that.
If you do discrete dilutions (with ever increasing buffer volumes), the results will be more precise and more reliable. Lets just say, your pipet has a mean error of 1% (stacking, for serial dilution), your larger volumetric flask has a mean error of some 0.4%. Don't just take the manufacturer precisions of the pipets into consideration, to these you also have to apply the deviations that come from the person using the instrument, which can vary widely among users and the consumables.
The correct way to prepare standards (and sample replicates) is by preparing each from the very start, independently, not by dilution. This will result in your standard curve representing your actual accuracy over the whole process. In addition you do multiple independent replicates of your sample (not technical replicates, which are simply measuring multiple aliquots of the same solution in order to compensate for the imprecision of your method of analysis). Which in a life science environment means doing at least three distinct cultivations and therefore extractions and analyses, plus technical replicates. The work at your feet rapidly gets out of hand if you want to work diligently.
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