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I have a Peptide A (mw = 5074Da), the table below showed measured concentration by Qubit from 5uM up to 80uM solution. All under 20uL solution.

enter image description here

Below is the standard plot for Peptide A (not sure if it's relevant or not):

enter image description here

Let's say I have another Peptide B (mw = 3102Da), how can we calculate the expected Qubit read of Peptide B based on Peptide A (just by calculation)?

For example just for 1 node: 20uM.

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    $\begingroup$ It does not seem to me to be linear. $\endgroup$
    – Poutnik
    May 24, 2022 at 6:17
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    $\begingroup$ I mean there is consistent bending down and that questions justification of linear approximation. $\endgroup$
    – Poutnik
    May 24, 2022 at 6:34
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    $\begingroup$ What is "nodes"? Please add some clarification on the concentration units, as these are orders of magnitude different (measured vs nominal). If you read the manual you'll see that the measured concentration can vary ~12% between proteins, but this will apply to peptides only if the functional principle of the assay still applies. I would think it's ok. $\endgroup$
    – Buck Thorn
    May 24, 2022 at 9:57
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    $\begingroup$ science.smith.edu/cmbs/wp-content/uploads/sites/36/2015/09/… $\endgroup$
    – Buck Thorn
    May 24, 2022 at 9:57
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    $\begingroup$ @BuckThorn Thanks so much for your reply. Nodes is the concentration in the peptide solution I put in the tube (before adding the working solution) to be measured with Qubit. $\endgroup$ May 24, 2022 at 11:45

1 Answer 1

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If knowing the concentration of peptide B is important and you have pure peptide B available, you should measure standards of known concentrations of peptide B in the assay.

The assay works through a dye that has little fluorescence in solution and a lot when bound to proteins. The mechanism of binding is not known or not divulged. While there is a claim that there is little variation of mass of protein sample vs. fluorescence signal, the data presented by the company uses larger globular proteins.

We don't know what the amino acid composition or the structure of your peptides are. We also don't know whether the assay works under native or denaturing conditions (the assay buffer has a pH between 6 and 8, but there is no information about its composition).

If knowing the concentration of peptide B is not so important, you could assume that the same mass of peptide will have the same fluorescent signal. Or you could use the assay to get a relative concentration if the absolute concentration is unimportant.

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