scinco Mega-800 - Protein;Dna Determination Methods; Gain Access to Dna;Protein Analysis

54 Mega-800 SCINCO
Turn on the MEGA-800 UV-VIS Spectrophotometer. After 30mins warm-up, press in the case
6.1.3 Protein/DNA determination methods
① Absorption method at 280nm and 260nm
DNA&RNA absorbs the UV light intensely. Its absorbance ability is 10 times as intense as
protein at 280nm. The absorption of DNA&RNA at 260nm is more intense. The extinction
coefficient at 260nm is twice as much as that of at 280nm. But for protein, the absorption
value at 280nm is larger than that at 260nm.
Because the protein absorpiton value and DNA&RNA absorption value are different, we
can judge the ratio of protein and DNA&RNA, for example:
● Pure protein absorbance ratio: A280/A260  1.8
● Mixed solution of protein and DNA&RNA: A280/A260  1.8～0.5
● Pure DNA&RNA absorbance ratio: A280/A260  0.5
Method:
As for the mixed solution of protein and DNA, we can determine their concentration values by
measuring their absorption values at the wavelength of 260nm and 280nm. The formulas is shown as
below:
Protein concentration ＝ 1552×A280－757.3×A260 (ug/ml) …………（6-1）
DNA concentration ＝ 62.9×A260－36.0×A280 (ug/ml)
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1
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………（6-2）
② Absorption method at 230nm and 260n
The peptide bond of protein has its maximum absorption value at 238nm, and we take A230 as an
approximate value. DNA&RNA also has intense absorption at 230nm. So we can determine A230 and
A260 with the fixed solution of protein and DNA.
Method:
We can determine the concentration values of protein and DNA by measuring their absorption values at
the wavelength of 230nm and 260nm. The formulas are shown as below:
Protein concentration ＝ 183.0×A230－75.8×A260 (ug/ml)……………...（6-3）
DNA concentration ＝ 49.1×A260－3.48×A230 (ug/ml)………………….（6-4）
The coefficients above need to be amende according to different situations.
Comparing with absorption method at 280nm and 260nm, the absorption method at 230nm and 260nm
is more accurate. But it is more easily interfered.
There is no absorbance at the wavelength of 320nm, so background correction at 320nm can be
preformed.
6.2 Gain access to DNA/Protein analysis
On the instrument main menu, press key to select the DNA/Protein mode. The main menu of
quantitative measurement will be displayed as shown in Fig. 6.1, press to return to the
instrument main menu.
【*1】Note:
1. The coefficients need to be amended according to different situations.
2.The method is easily interfered, such as the NaCL and the solution with conjugateddoublebond.