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BSA Standard Curve Dilution (BCA Kit)


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6 replies to this topic

#1 Nose

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Posted 11 June 2010 - 01:59 PM

Hi,

the Pierce BCA Kit comes already with Albumin Standard Ampules, 2 mg/ml, diluted in a solution of 0.9% saline and 0.05% sodium azide that are used for the Preparation of Diluted Albumin Standards.
As you know, the standard should be diluted in the same buffer as the sample. Letīs say the sample is diluted in RPMI or any other medium - you are advised to use the same medium for your diluted albumin standards.
My problem is: When you do a series of a 1:2 dilution, there will be always 50% salt solution and 50 % medium for every standard except the 2mg/ml beginning standard that has 100 % salt solution (ok, for the lower standards the salt solution will be diluted out but something is still there). On the one hand, there will be less background signal measured (assumed that there is one) compared to the samples that are in 100% medium. On the other hand, you have to subtract the blank (medium) from your standards that is nonsense for your highest standard (2 mg/ml) because it isnīt diluted in medium.

Hope you get my point!

Edited by Nose, 11 June 2010 - 02:01 PM.


#2 Gerard

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Posted 12 June 2010 - 06:24 AM

I dilute the standard with Saline 0.9% and have no concern about the matrix of my samples because I assume the the fact that in the samples a mix of protein are present and not only albumin as in the standard this will have more influence on my results.
Ockham's razor
Pluralitas non est ponenda sine necessitate
-- "You must assume no plural without necessity".

#3 medchemgirl

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Posted 14 June 2010 - 10:25 AM

What you mean by saline, PBS?????


I dilute the standard with Saline 0.9% and have no concern about the matrix of my samples because I assume the the fact that in the samples a mix of protein are present and not only albumin as in the standard this will have more influence on my results.



#4 Gerard

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Posted 14 June 2010 - 11:52 AM

I mean just NaCL 9g/l but PBS will do to.
Ockham's razor
Pluralitas non est ponenda sine necessitate
-- "You must assume no plural without necessity".

#5 medchemgirl

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Posted 14 June 2010 - 12:44 PM

I see, thanks!

I mean just NaCL 9g/l but PBS will do to.



#6 rachelhauser

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Posted 05 March 2011 - 12:28 PM

I see, thanks!

I mean just NaCL 9g/l but PBS will do to.


Why not just BUY a BSA standard (pretty cheap), weigh out and prepare a 2mg/mL solution and make your own standard curve? That's what I do, no problems with any interferences, since it's only BSA in MQ water..
"Live Long and Prosper".

#7 Kaioshin

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Posted 12 March 2011 - 06:17 PM


I see, thanks!

I mean just NaCL 9g/l but PBS will do to.


Why not just BUY a BSA standard (pretty cheap), weigh out and prepare a 2mg/mL solution and make your own standard curve? That's what I do, no problems with any interferences, since it's only BSA in MQ water..


My advisor is adamant that BSA standards be made the correct concentation through spectrophotometric measurment and calculation using the extinction coefficient. Just weighing the "correct" amount of BSA and dissolving in solution doesn't give 100% the correct concentration. Just something to throw out there.




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