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Topic: Dilution factor when calculating BSA concentration  (Read 5569 times)

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Offline ckretai

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Dilution factor when calculating BSA concentration
« on: October 16, 2017, 04:52:26 AM »
I'm a little bit confused.. do I need to apply a dilution factor in this scenario? And if so did I determine it correctly?

Using stock BSA solution of unknown concentration, I prepared a diluted BSA solution. 50μL of stock BSA and 4950μL of distilled water. I then prepared a cuvette containing 1mL of my dilute BSA solution and 2mL of Bradford reagent. The absorbance was measured and concentration of BSA was calculated to be 42.42 μg/mL (by solving for x from the equation of the line of my known protein calibration curves.)

To calculate the original protein concentration of the stock BSA solution, does this make sense?
1st dilution (BSA in water) = 5000μL/50μL = 100 fold
2nd dilution (BSA in Bradford) = 3mL/1mL = 3 fold....
100*3 = total dilution factor of 300

I know that Cfinal = Cinitial/dilution factor... or Cinitial = Cfinal*dilution factor
Thus Cinitial = (42.42 μg/mL)(300) = 12726.9μg/mL.

If this is right, this is a weird number to me. If I have in fact done this right, should I be converting it to a better unit? I feel like mg/mL makes more sense to look at.

Offline Borek

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Re: Dilution factor when calculating BSA concentration
« Reply #1 on: October 16, 2017, 06:14:56 AM »
At first sight I see nothing wrong in your calculations. Yes, mg/mL seems to be a bit better selection of units here.

Not that there is anything intrinsically wrong with using μg/mL for such a result. Actually it some situations it can be better. For example if you report series of measurements and all other results are below 1000 μg/mL and are reported using μg/mL, it will be better to write 12726.9 μg/mL than 12.73 mg/mL, as the latter could be easily overlooked as being much higher by someone reading the data.
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Offline Babcock_Hall

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Re: Dilution factor when calculating BSA concentration
« Reply #2 on: October 17, 2017, 05:40:04 PM »
My comment is not about the math.  Your ratio of protein solution to Bradford reagent is unusually large in my experience.  Is that from a protocol?

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