# concentation calculations - (Jun/19/2013 )

I have received 3 peptides labeled with FAM (5,6 carboxyfluorescein) from GenScript.
1) Fam-Tat 1mg, MW=1979.22 g/mol
2) Fam-Q33 1mg MW=4269.78 g/mol
3) Fam-E12 1mg MW=1814.95 g/mol

I need to use 50 uM concentrations. I calculated it in two ways and (sic!) I got 2 completely different results, could anybody help with that?

Here are the details of my calculations:
I. Assuming that I got exactly what the company claimed to send so 1mg.
II. Dissolving the whole amount which I received in 1ml and measuring the absorbance at 495 nm with the spectrophotometer

-> I. 1) Fam-Tat 1mg, MW=1979.22 g/mol
1L - 1979.22 g
1L - 1979.22 x 10^3 mg - 1M
1mg - (1/1979.22 x1000) - 0.5 x 10^6 M = 0.5 uM

1000 ml - 0.5 uM
1 ml -0.5 x 1000 uM = 500 M

500 x X = 50 x 100
X= 10 ul (of the stock peptide)
to dissolve in 90 ul of PBS

-> II. 1) Fam-Tat- spectrophotometer Shimadzu,
Absorbance at 495nm A(495) = 0.643, dilution factor =40, Extinction coefficient of FAM = 67636.4
A = e b c
(0.643) x (40) = 67636.4 x c
c = 0,0003803 M = 380 uM
(3.803 x 10^-4)x V = (600 x 10^-6) x (50 x 10^-6)
V = 78.9 ul (of the 40 fold dilution)
to dissolve in 521 ul of PBS

-nusphd-

as i posted in answer to the same question in the biotechniques forum:

"i would give more weight to method 1. there are too many things that could influence absorbance readings to trust method 2 with only one dilution read."

-mdfenko-