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### Topic: Protein assay - determining amount of protein from standard curve  (Read 731 times)

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#### yogurtspoon

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##### Protein assay - determining amount of protein from standard curve
« on: September 18, 2020, 02:56:47 PM »
For my lab, we did a piers 680 protein assay using a lysozyme stock solution. They had us make three solutions of unknown concentration by preparing different dilution ratios using a stock solution of an unknown protein, ex. for mine I did 1:1, 1:5, and 1:10 dilutions.

I already made the standard curve and got the concentrations for each.
1:0 = 1478.2 micrograms/mL
1:4 = 294.8 ""
1:9 = 140.2 ""
I thought this seemed like decent data since the 10-fold dilution is ~1/10th of the undiluted solution, and the 5-fold dilution is about 1/5th of the undiluted solution. But I'm confused about how to find the amount of protein in micrograms?

I thought I was supposed to just multiply by the volume of the solution (1.6 mL). Then that would be
1:0 = 2379.52 micrograms
1:5 = 471.52 ""
1:10 = 224.32 ""
I'm confused because these values are bigger than the amounts in the lysozyme standards. Specifically, the 1:1 and 1:4 are outside of the standards' range (the standards had between 2.5 - 200 micrograms of lysozyme). The 1:9 is within, but since its concentration falls between 2 standards, shouldn't the unknown's amount of protein (224.32) also fall between that of those 2 standards (12.5 - 25 micrograms)?

My TA also mentioned multiplying by the dilution factor to get the right amount of protein. But that would just make the numbers even larger so Idk if I'm remembering right. Should I be multiplying by 1/5 and 1/10 instead of multiplying by 5 and 10? He said this calculation was to "compensate" for the dilution but I didn't really get what he meant when he explained it.

Not sure if this is necessary info – but to clarify, the unknown tubes were prepared by adding the unknown stock soln, dH2O as a buffer, and then 1.5mL piers protein reagent for all tubes. So for the 1:5, it was 20 microliters unknown stock, 80 microliters dH2O, 1.5 mL piers protein reagent.

Thanks in advance for any help.
« Last Edit: September 18, 2020, 05:49:44 PM by yogurtspoon »

#### mjc123

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##### Re: Protein assay - determining amount of protein from standard curve
« Reply #1 on: September 18, 2020, 05:05:05 PM »
I'm not familiar with this method, but how was the calibration curve constructed? For example, does "1:1 = 1478.2 ug/mL" mean that this sample has an absorbance corresponding to a standard of 1478.2 ug/mL prepared in the same way, i.e. 100 uL of standard of this concentration plus 1.5 mL piers reagent? If so, that is the concentration of the 100 uL sample, not the 1.6 mL solution. If you multiply your found concentrations by 0.1 mL, they seem to give amounts more in line with your expectations.

By the way, you need to sort out your dilution terminology. I assume, from the ratios of the concentrations, that your samples were:
Undiluted, i.e. 100 uL stock, no added water - that's 1:0 sample:diluent.
5-fold diluted, i.e. 20 uL stock, 80 uL water - that's 1:4
10-fold diluted, i.e. 10 uL stock, 90 uL water - that's 1:9
Adding 1 part sample to n-1 parts diluent (1:n-1) gives you an n-fold dilution. Calling the undiluted sample (if that's what it was) 1:1 is particularly misleading.

#### yogurtspoon

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##### Re: Protein assay - determining amount of protein from standard curve
« Reply #2 on: September 18, 2020, 05:48:40 PM »
To make the curve, we prepared 8 lysozyme solutions with increasing concentration. (The solns contained lysozyme stock and water, for an added volume of .1mL; and then 1.5 mL of Piers 660 protein reagent, which I think is needed to produce the color change.) We measured their abosrbances at 660 nm, then plotted them as absorbance vs. concentration and added a linear regression line. Then took the absorbances of the 3 unknown solutions, and plugged them into the regression equation to solve for their concentration. The absorbances were prepared in the same method as the standards (unknown protein stock + water for added volume of .1mL; 1.5mL reagent). But the unknowns don't have the same combination of protein stock & water as the standards, except for the 1:0* dilution which matches my most concentrated standard. I'm not sure if what you said still applies?

*For the dilutent terminology, thanks for the correction! I did mean 1:0, it's undiluted - I'll edit my original post on that one. My TA also mentioned the 1:4 thing, but said that if it was easier for us to understand then we could refer to it as 1(part):5(whole) instead. I thought the terms were interchangeable for most, but maybe not! Sorry I made it unecessarily confusing there.

#### mjc123

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##### Re: Protein assay - determining amount of protein from standard curve
« Reply #3 on: September 20, 2020, 07:31:27 AM »
Just to check, when you say you plotted the absorbance vs. concentration, was the concentration value you used that of the original lysozyme solution (a certain amount of protein in 0.1 mL) or that of the final sample (the same amount of protein in 1.6 mL)?

And please do not edit your original post after it's been commented on; that can make the comments unintelligible. Make corrections in another post.

#### Babcock_Hall

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##### Re: Protein assay - determining amount of protein from standard curve
« Reply #4 on: September 20, 2020, 10:12:39 AM »
I think that mjc123 asked the right question.  To address another issue, it is possible that an unknown could have a slightly higher concentration than any of the standards.  If it did, what inferences would you draw?

#### yogurtspoon

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##### Re: Protein assay - determining amount of protein from standard curve
« Reply #5 on: September 20, 2020, 10:34:54 AM »
mjc123: We plotted absorbances vs. the concentrations of the final samples (same amount of protein in 1.6 mL). My bad on the editing thing as well, I'm new on this forum and thought reddit etiquette applied.

Babcock_Hall: I'm honestly not sure. The most concentrated unknown should just about match my most concentrated standard, so wouldn't deviations be caused by my own imprecisions when I prepared the solutions?

#### Babcock_Hall

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##### Re: Protein assay - determining amount of protein from standard curve
« Reply #6 on: September 21, 2020, 10:19:14 AM »
If had an unknown protein solution and had no idea of what its concentration was, I might choose a dilution protocol for it that caused it to have a larger concentration than the highest standard.  For that matter I might just as easily pick a dilution factor that caused it to have a lower concentration than the lowest standard.