How to use Nycodenz?
How to use Nycodenz?
Nycodenz (iohexol) has a molecular weight of 821 grams per mol.
Its density is 2.1 g/ml.
Solutions of over 80 % (w/v) can be prepared.
Nycodenz is stable to heat and may be autoclaved, stability to autociaving is enhanced
by the addition of small amounts of Tris and EDTA.
Nycodenz in solid form is stable for aperiod of 8 years when stored
at room temperature and protected from light.
Gradients of Nycodenz can be generated in the following ways:
- Formed in situ by centrifugation (selt-forming gradients).
- Layering solutions of the desired concentration into an
appropriate centrifuge tube and allowing the solutions to diffuse.
Using Nycodenz® isotonic solution gradients can be simply
prepared within 45 minutes (see next section).
- Freezing and thawing.
- Gradient mixers.
How to prepare?
Am Iso-osmotic solution of Nycodenz® which contains 27.6% (w/v)
Nycodenz® (density = 1.15 g/ml) in buffered medium.
This solution may be diluted to desired concentration by using a buffered
diluent containing either sucrose or NaCI as osmotic balancer.
The composition of these diluents are as folIows:
0.75 g NaCI or 7.45 g sucrose dissolved in 100 ml 5 mmol/l Tris-HCI (pH 7.5) containing 3 mmol/l KCI and 0.3 mmol/l CaNa2 EDTA.
The relationship between density and refractive index (η) can be
NaCI diluent Sucrose diluent
Density = 3.287 η - 3.383 Density = 3.410 η - 3.555
COMPATABILITY WITH SOME WIDELY USED ASSAYS
Nycodenz® does not interfere with the orcinol and diphenylamine
reactions for estimation of nucleic acids, nor with the very sensitive
dyebinding assays for protein and DNA. Polysaccharides and
sugars can be determined in the presence of Nycodenz® using the
assay. Fluorimetric assays of nucleic acid and
proteins can also be carried out in the presence of Nycodenz®.
Nycodenz® does not interfere with most assays for the marker
enzymes of subcellular components, also most commercial
scintillants are compatible with Nycodenz®.
REMOVAL OF NYCODENZ® FROM SAMPLES
Nycodenz® can be removed tram samples by dialysis, ultrafiltration
and gel filtration. Cells, subcellular organelles and other particulate
matter can be separated from Nycodenz® by centrifugation without
the risk of contaminating the pellet with Nycodenz®.
Nycodenz® is readily soluble in both aeidic and ethanolic media.
Thus in a number of cases samples can be isolated free of
Nycodenz® by precipitating the sampIe with trichloroacetic acid or
Nycodenz® prod. no. 1002424 1 x 500 g