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Mr_Saturn512's avatar

How do I figure out the dilution factor for this equation?

Asked by Mr_Saturn512 (346 points ) 2 months ago

Long story short, I measured A280 of a protein with unknown concentration in mg/mL. I figured out the exact concentration doing a standard curve and regression line and yada yada yada. So that’s good.

But now, for the sake of seeing if this OTHER method still gives us the same numbers as what I did with the standard curves, I have to do the following equation to figure out the mg/mL concentration of the protein:

(absA280) (dilution factor)/Extinction coefficient of protein = concentration of protein

The extinction coefficient of this protein I’m working with is 51,300.

I’m just going to throw a random abs I got, so for example – .306.

The mg/mL I got using the standard curve equation is 2.52 mg/mL. Which is good. That was the 2.5 mg/mL standard.

But now that I’m using this equation, I can’t seem to get a number close to 2.52 mg/mL. I resorted to throwing numbers to plug in as the dilution factor as guesstimates, but I still got nothing remotely close.

So I really have no idea what they mean by “dilution factor” in terms of this equation. They don’t show an example, which is annoying.

NOTE: And yes – I tried solving for the dilution factor, treating it as X and I got some number that doesn’t make sense (like 422,47 blah blah)

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2 Answers

Mr_Saturn512's avatar

OKAY. So I figured out how to get the dilution factor (total volume divided by the aliquoted volume) and that’s all fine and dandy.

However, now I still don’t get an appropriate answer.

Let’s take my 2.0 mg/mL sample

I made it by diluting 8mL from 2.5 mg/mL into 2mL of water.

Therefore, the dilution factor should be 1.25 (10 divided by 8).

The A280 value I got for this standard was .240

So: .240(1.25)/51,300 = .0000005847 (aka – WTF) It won’t make a difference playing around with the decimal point because it’s still going to be 5847, which is not 1.98 mg/mL (my calculated result from the regression line) or anywhere close to 2.0 mg/mL

Mr_Saturn512's avatar

So I figured out what’s the matter.

This extinction coefficient thing I’m using is in mols/Liter. We want mg/mL. I’m not entirely sure how to convert, because I need to do some funky stuff with the molecular weight of this protein, right? I may just ask another question.

OR. I can just convert the mg/mL into mol/Liter. . .

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