Biology Reference
In-Depth Information
Estimation of Iron Export from Cells
Cell lines expressing different PrP forms were radiolabeled with
59
FeCl
3
-citrate com-
plex as above. Cell surface bound iron was chelated with three washes of PBS supple-
mented with DFO (100 μM) and the cells were chased in complete medium for dif-
ferent time periods. A 50 μl aliquot of the medium was retrieved at each time point
and counted in a γ-counter. After 16 hr, cells were lysed and cell associated iron was
measured in a gamma counter.
Estimation of Ferroxidase Activity of Recombinant PrP
Ferroxidase activity of PrP was measured by the published colorimetric method us-
ing 3-(2-pyridyl)-5,6-bis(2-(5furylsulfonic acid))-1,2,4-triazine that forms a colored
Fe
2+
complex with ferrous iron (44) with the following modifications: Reagent A:
0,45 mol/l sodium acetate, pH 5.8, reagent B:130 mmol/l thiourea, 367 μM/l Fe(NH
4
)
(SO
4
)
2
×6H
2
O, reagent C (chromogen): 18 mmol/l 3-(2-pyridyl)-5,6-bis(2-(5-furylsul-
fonic acid))-1,2,4-triazine in 0.01 M Tris pH 7.0. Each sample contained either 1 μl
of water or 1 μl of 300 μM CuSO
4
, 6 μL of the sample (undiluted human plasma, hu-
man serum albumin 70 g/l (Sigma A1653-5G) in PBS or recombinant prion protein
(0.6 μg/ml) and 820 μl of reagent A. Multichannel pipette (Finnpipette) was used
for therapid addition of the reagent B (substrate) to minimize the time difference in
sample processing. Sample quadruplicates were incubated at 37°C for 4 min. Unoxi-
dized Fe
2+
was reacted with 60 μl of chromogen solution (reagent C) and absorbance
was measured at 600 nm with Smart Spec Plus (BioRad) spectrophotometer. Copper
was added to provide two copper ions per PrP molecule, and was also added to human
albumin and plasma samples. The amount of PrP protein in PrP-containing samples
(3.6 μg/sample) roughly corresponds to a known amount of ceruloplasmin in 6 μl of
undiluted human plasma. As a control, purified 99% human serum albumin was used
(70 g/l in PBS) to mimic the total protein concentration in plasma. As a blank samples
were supplemented with 6 μl of de-ionized water instead of albumin solution, plasma
or recombinant PrP solution.
RNA Isolation and Northern Blotting
The M17 and WT cells cultured in the absence or presence of 0.1 mM FAC for 24
hr were washed with cold PBS, trypsinized, and collected in 1.5 ml eppendorf tubes.
Total RNA was isolated by using SV total RNA isolation kit (Promega, Madison, WI)
and quantified. 15 μg of total RNA was fractionated on 0.8% formaldehyde agarose
gel followed by blotting to positively charged Nylon membranes (Roche diagnostics).
Membranes were hybridized with DIG-labeled PrP or β-actin probes and binding was
detected by the CSPD reagent.
STATISTICAL ANALYSIS
Data are presented as the mean ± SEM values. Statistical evaluation of the data was
performed by using Students t-test (unpaired).
