Biomedical Engineering Reference
In-Depth Information
4.4.1 Preparation of Tissue Powder (Liver Powder)
A 0mM phosphate buffer, pH 6.5
Solutions/Reagents
acetone
diethylether
Wash pig liver with water and remove as much blood as possible.
Homogenize chopped pig liver in a blendor with the same volume
of Soln. A and cool the homogenate to 0
◦
C.Pourslowlytheho-
mogenate to 10 vol. of acetone cooled to −15
◦
C. Allow to settle
at −15
◦
C and filter the sediment on a Büchner funnel. Wash the
tissue twice with three volumes of cold acetone, followed by a wash
with cold diethylether. Spread the dehydrated tissue on filter paper,
grind up, allow the solvent to evaporate and store it in a desicca-
tor containing anhydrous calcium chloride at 4
◦
C.Thewhiteliver
powder remains stable for months.
References
Kleber H-P, Schlee D, Schöpp W (1997) Biochemisches Praktikum. 5th ed.,
G. Fischer, Jena, p 24
4.5 Preparation of Egg Yolk IgY Fraction
A : 0mM Tr i s
·
HCl, 0.5 M NaCl, pH 7.5
Solutions/Reagents
B 10% (w/v) dextransulfate in A
C1M CaCl
2
D .5M EDTA, disodium salt, pH 7.5
E
saturated ammonium sulfate solution (see Protocol 4.3)
solid ammonium sulfate
Separate yolks of chicken eggs from egg white and discard egg
white. Wash the yolks carefully with water to remove adhering egg
white.Suspendtheyolksin5vol. Soln. A by vigorous stirring.
Precipitate lipids and lipoproteins by addition of 6 ml Soln. B and
15 ml Soln. C per 100 ml yolk suspension. Stir at RT for 30 - 60 min
and spin at 5000
g for 10 min. Wash the pellet with a small volume
of Soln. A (about 20 ml per yolk) and centrifuge again. Combine
the supernatants and filter through a paper filter. Add Soln. D to
the clear filtrate to a final concentration of 30 mM EDTA.
Add 24.3 g solid ammonium sulfate per 100 ml EDTA-filtrate
(yields a 40% saturation) and stir at 4
◦
C for 30 min.Collectthe
precipitate by centrifugation. Wash the pellet once with 30% satu-
rated ammonium sulfate (3 vol. Soln. E + 7 vol. ddH
2
O), spin down
again and dissolve the precipitated IgY in a small volume of Soln. A
(about 1/10 of the original yolk volume). Dialyze against TBS and
determine the IgY content spectrophotometrically.
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