With the aid of spectroscopy, the quantitative analysis of nucleic acids
and proteins has established itself as a routine method in many laboratories.
It includes absorption measurements in the ultraviolet and in the visibility
range. Proteins are measured (directly) at 280 nm, nucleic acids at 260
nm and colorimetric protein determination is carried out in the range from
550 to 600 nm. The BioPhotometer offers the following pre-installed test
1. Nucleic acid determination
DNA, RNA, oligonucleotides and even mononucleotides can be measured directly
in aqueous solutions in a diluted or undiluted form. Aqueous buffers with
low ion concentrations (e.g. TE buffer) are ideal for this method. The concentration
is determined by measuring at 260 nm against blank and then evaluating via
factor. Normally, the user has to calculate the concentration of the measured
sample using according factors. In contrast, the BioPhotometer can change
these factors easily and will do all necessary calculations.
The absorption of 1 OD (A) is equivalent to approximately 50 g/ml dsDNA,
approximately 33 g/ml ssDNA, 40 g/ml RNA or approximately 30 g/ml for
oligonucleotides. Purity determination of DNA Interference by contaminants
can be recognized by the calculation of ratio. The ratio A260
is used to estimate the purity of nucleic acid, since proteins absorb at
280 nm. Pure DNA should have a ratio of approximately 1.8, whereas pure
RNA should give a value of approximately 2.0. Absorption at 230 nm reflects
contamination of the sample by substances such as carbohydrates, peptides,
phenols or aromatic compounds. In the case of pure samples, the rat
should be approximately 2.2.
2. Protein determination
The protein content of a preparation can be determined on the basis of various
different analytical procedures. Evaluation can be carried out via factor
or via a calibration curve, with up to ten standards in the BioPhotometer.
Absorption measurement at 280 nm (A280)
method may be used with in concentrations of up to approximately
4 mg/ml (3.0 A). This method is simple and rapid, but may be disturbed by
the parallel absorption of non-proteins (e.g. DNA). Unlike the colorimetric
process, this method is less sensitive and requires higher protein concentrations
and should thus be used with pure protein solutions. In addition to the
direct absorbance display, evaluation is possible with the BioPhotometer
via the Warburg formula or via standard.
Colorimetric determination (dye tests)
Protein samples often consist of a complex mixture of many different proteins.
The quantitative detection of the protein content is usually achieved on
the basis of the reactions shown by functional groups of the proteins to
dye-forming reagents. The intensity of the dye correlates directly with
the concentration of the reacting groups and can be measured exactly.
Lowry assay 595nm
Specialist literature contains a multitude of modifications for the Lowry
assay. The principal target is to reduce the high susceptibility to interference.
In comparison to the pure Biuret assay, the sensitivity of this assay has
greatly increased. However, the Lowry method is adversely affected by a
wide range of
non-proteins. Additives such as EDTA, ammonia sulfate or Triton
X-100 in particular are incompatible with the test.
Bicinchoninine acid assay 562nm (BCA)
This test represents a highly regarded alternative to the Lowry assay. It
is easier to carry out and sensitivity can be varied using different temperatures.
Furthermore, the dye complex is very stable. However, this test is highly
susceptible to interference, although on the positive side, its insensitivity
to detergents is similar to that of the Lowry method.
Bradford assay 595nm
This method is twice as sensitive as the Lowry or BCA test and is thus the
most sensitive quantitative dye assay. It is the easiest to handle and most
rapid method and has the additional advantage that a series of reducing
substances (e.g. DTT and mercaptoethanol), which interfere with the Lowry
or BCA test, have no adverse effect on results.However, it is sensitive
to detergents. The main disadvantage is that identical amounts of different
standard proteins can cause considerable differences in the resulting absorption
3. Bacterial cell density
The density of bacterial suspensions may be measured photometrically at
595 nm without dyes having to be added. This applies e.g. to the preparation
of competent cells (i.e. cells, which are able to absorb plasmid DNA), that
must be in a specific phase of growth.
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