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I have two peptide A and B. And I would like to know the solubility level of these peptide in certain solute. For that purpose I use Qubit Protein Assay.

The Qubit readout I get is this:

enter image description here

Note that the Qubit Read Concentration and Fluorescence of Peptide B is lower than Peptide A.

As far as I understand, we use fluorescence to quantify the peptide (so it's equivalent to absorbance). High fluorescence means lower solubility.

Just by looking at fluorescence value I'm inclined to say that Peptide B has lower solubility than Peptide A. But my worry is that the Qubit Read Concentration are different for both. I wonder if this case will make the two peptide fluorescence incomparable?

If not, what is the better strategy to deal with it?


Update (based on @Karsten comment)

Here is the standard curve for Peptide A and B. Can we still say that Peptide B is less soluble then Peptide A?

enter image description here

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  • $\begingroup$ Please note that standard way of emphasizing in Markdown is using asterisks: *<emphasized text>* (<emphasized text>). Backquotes are only used for inline code: `<code>`. $\endgroup$
    – andselisk
    Commented May 26, 2022 at 17:37
  • $\begingroup$ Try plotting an overlay of concentration of the peptides in g/L vs determined (Qubit) concentration. In other words, multiply conc(uM) by the molecular weight. I suspect the curves will overlap pretty well. $\endgroup$
    – Buck Thorn
    Commented May 27, 2022 at 9:45
  • $\begingroup$ This data might tell you if your peptides self-associate, but not the solubility. $\endgroup$
    – Buck Thorn
    Commented May 27, 2022 at 9:46
  • $\begingroup$ By the way please edit the title of your post to better reflect its contents. $\endgroup$
    – Buck Thorn
    Commented May 27, 2022 at 9:48

1 Answer 1

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If these two peptides are the same as those in this question, you would expect a higher reading and higher estimate of mass for solutions of A compared to solutions of B of the same concentration. This is because for every molecule of peptide A, you have a larger mass and a larger chance of fluorescent dye attachment compared to B (the molar mass ratio is about 5:3).

The question mentions solubility limit. While the concentrations are not linear with the signal, it does not seem that there is a ceiling to the signal at higher concentrations, so maybe the solubility limit is not reached. We would need a bit more context to understand what the experimental setup is to fully address that aspect.

Here is a sketch of what you would expect if you dissolve different masses of peptide in a given volume, spin down and measure the supernatant:

enter image description here

The solutions that are not saturated should show a signal roughly linear to the mass added. At the same amount (nanomoles), you would expect peptide A to have a higher signal because there are more amino acids to interact with the fluorescent dye. Once you reach the solubility limit (highlighted in the spreadsheet), some of the material precipitates, so there is a cap on the fluorescent signal. To figure out the solubility, extrapolate the signal until you reach the signal of the saturated solutions, and read off the amount and calculate the concentration. (I could have used concentrations and mass concentrations in the spreadsheet, with the same conclusion).

[OP] High fluorescence means lower solubility.

That does not make any sense to me. If the solubility is higher, there will be a higher concentration in a saturated solution, and the fluorescence signal should be higher.

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    $\begingroup$ thanks for your answer. So you meant the Qubit read cannot be used to determine which peptide has better solubility. Am I right? How about calculating the fold change of Qubit read with initial concentration/mass before creating the solution? What other context we can give you so that you can suggest us the experimental setup? $\endgroup$ Commented May 25, 2022 at 22:20
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    $\begingroup$ The easiest experiment is to make a saturated solution, spin it down and measure the concentration of the supernatant. This gives you a rough idea of the solubility (mass per volume). In you experiments, did you see a pellet after centrifugation? $\endgroup$
    – Karsten
    Commented May 26, 2022 at 1:13
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    $\begingroup$ how do you measure the concentration of supernatant? Yes I can see the pellet after centrifugation. $\endgroup$ Commented May 26, 2022 at 1:36
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    $\begingroup$ In the Qubit. But maybe I just don't understand the data you are presenting. If you have known amounts of peptide, you can get a calibration curve for solutions that are not saturated. Then, you measure the supernatant of the saturated solution (diluting it if it is outside the range of the standard curve). This will give you a good estimate of the solubility. $\endgroup$
    – Karsten
    Commented May 26, 2022 at 2:59
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    $\begingroup$ The supernatant of a saturated solution has an unknown concentration (because some material is in the pellet). Not saturated solutions have a known concentration (everything is in solution), so you can use those to make the standard curve. $\endgroup$
    – Karsten
    Commented May 26, 2022 at 17:31

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