Supporting data
Standardization of Capillary immuno-electrophoresis (CIE) for PrPC detection
The sensitivity and specificity of the method depends on the number of free antigen binding
sites on the antibody and concentration (mg/ml) of the peptide. Therefore, too high
concentration of the antibody as compared to the PrPC and peptide concentrations decreases
the chance of competition between the PrPC and fluorescent peptide leading to most of the
sites on antibody remain free. Thus, in order to measure the differential expression of PrPC in
the tissues from different groups, the precise standardization of the antibody dilution and free
FITC-peptide concentration was required.
The determination of the optimal dilutions of the peptide and purified antibody was done by
two different binding assays. First binding assay was performed by the preparation of
antibody dilutions in a range from 1/50 to 1/250 with a constant concentration of peptide
(1/10,000 of the stock (0.5 mg/ml)) (Fig. 1S.A).
Further, a second binding assay was performed by the preparation of peptide dilutions in a
range from 1/25,000 to 1/100,000 with a constant antibody dilution (1/300). The fluorescence
absorption in both binding assays was decreased linearly (Fig. 1S.A and 1S.B).
The difference between the values of RFUs of free FITC-peptide and RFUs of the peptide
after binding to antibody (free peptide + antibody) were required to be sufficiently high to
cover a broad range of protein expression. Finally, we used an antibody dilution (1/300) with
a peptide concentration (1/33000 dilution of the stock (0.5mg/ml)) for measuring the
regulation of PrPC expression.
As a confirmatory test of the standardized CIE based method, we showed the higher
expression of PrPC in the brain as compared to the liver (Fig. 3S), which was correlated with
previous reports of PrPC expression by Western blot technique [13].
Calculation of PrPC concentrations
The molecular mass of the peptide (DYEDRYYRE) is 1308.35 g/mol. So, the number of
peptide molecules in 1308.35 g of peptide was equivalent to 6.022 × 1023 and in 0.5 × 10-3
g/ml (concentration of the peptide) there were 2.3 × 1017 molecules per milliliter. As we
diluted the peptide concentrations to 1/33,000 times, therefore the actual number of molecules
in the used FITC-peptide dilution was 6.97 × 1012/ml.
As showed, there was a linear relationship between the peaks obtained after binding of
antibody with a range of dilutions against constant peptide concentration and also between
peptide with a range of dilutions against the constant antibody concentration (Fig. 1S.A and
1S.B). Therefore, the number of peptide molecules in the antibody peaks (FITC-peptide +
antibody) and PrPC peaks was calculated (FITC-peptide + antibody + tissue lysate) by
equating the area under the peaks of antibody and PrPC with the area under the peaks of the
free peptide (Supplementary table 1 and 2).
The difference between the number of peptide molecules of antibody control peaks and PrPC
peaks gave the number of PrPC molecules actually present in the 95 µl of the incubated
sample volumes.
Therefore, the concentration of PrPC in ng/ml was calculated by using the theoretical
molecular mass of un-glycosylated full length PrPC (27.90 KDa). Further, the values of PrPC
concentrations in pg/µg of the total protein were obtained by dividing the calculated PrPC
concentrations with the total protein concentrations of the samples used for the immunoassay
(Supplementary table 1 and 2).
Reproducibility
The reproducibility of the capillary immuno-electrophoresis was evaluated by applying
percentage inter-assay co-efficient of variance (CV%) to 3 independent set of experimental
replicates (Supplementary table 3 and Fig. 4S). The average CV% in the groups with
significant higher expression of PrPC in female than male (9 and 14 months) was 13.08. CV%
in 3 months group was high because the expression of PrPC in this group was close to the
detection limit of CIE and hence, non-significant results were obtained in this group. The
negative values in the 3 months group (data set 2, 3F and 3M) indicates that area under PrPC
peaks were more or less close to the area under antibody control peaks with random noise
fluctuations.
However, intra-assay coefficient of variance did not apply to our assay, because unlike ELISA
(with duplicates or triplicates), each sample was analyzed one at a time in the CE, followed by
washing of capillary with buffers. So, one set of biological replicates from 3, 9 and 14 months
(male and female) were run only once in one experiment, followed by replacement with a
fresh set of buffers for the second experiment. A long run of samples (i.e. including multiple
readings from each sample) with the same set of buffers may cause adsorption of the
fluorescent protein peptides to the inner surface of fused-silica capillary due to a known
limitation of capillary electrophoresis [23-26], which may lead to broadening of the peaks in
the last set of measurements.
Legends
Fig. 1S. Binding assays of antibody vs peptide: (A) shows the relative fluorescent units
(RFUs) obtained by the binding assay of FITC-peptide against different dilutions of PrPC
specific antibody. (B) Indicates the binding assay of PrPC specific antibody against different
dilutions of the FITC-peptide.
Fig. 2S. PrPC expression in the wild type compared to the PrPC knockout mouse liver:
shows the expression of PrPC in the wild type (peak 1) and PrPC knockout mice liver (peak
2). PrPC expression in PrPC knockout liver was equivalent to the antibody control (peak 3 antibody + FITC-peptide + 2% CHAPS buffer). Peak 4 represents the Free-FITC-peptide.
Relative Fluorescence Units (RFUs).
Fig. 3S. Electrophoretograms of PrPC expression by Capillary immuno-electrophoresis
(CIE): shows the expression of PrPC in the (A) brain and (B) liver of wild type mice. Peak 1
represents (Free FITC-peptide), peak 2 represents (Antibody + FITC-peptide + tissue lysate)
and peak 3 represents antibody control (antibody + FITC peptide + 2% CHAPS buffer). (C);
shows the quantification of PrPC expression by calculating their concentrations
(Supplementary table 2). Relative Fluorescence Units (RFUs).
Fig. 4S. PrPC expression (Reproducibility): PrPC expression from 3 datasets obtained by 3
independent set of experiments, shows the consistency in the expressional profile (3M - 3
month-old, 9M - 9 month-old and 14M - 14 month-old).