2 Materials
2.1 Capillary
Electrophoresis
Instrument’s Software
Every capillary electrophoresis instrument has its specific software
to retrieve the data.
2.2 Computer
The use of a programming language is necessary to process all the
data. For this project, Python has been chosen.
2.3 SPIKE
Package and Software
Availability
A Python package is available in https://spikedoc.bitbucket.io
[12, 13]. The software can be found in free access here: https://
github.com/lio3867/BiDop
3 Methods
All mentioned steps were performed with in-house Python scripts
using different packages, including SPIKE. This protocol is meant
to be applied to both electropherograms recorded for the reference
and the sample. The protein extract loaded on the reference and the
sample resins should come from the same expression source and in
practice from the same storage tube. This protocol also requires an
internal control added directly to the common storage tube, before
starting the experiment. This control is usually a soluble protein
with an adequate molecular weight, avoiding in particular any
overlap with the molecular weight of the protein of interest. The
peak intensity of the internal control protein (hereafter referred to
as the “internal control peak”) is used for normalization so that the
two electropherograms obtained from the same source tube can be
directly compared with the highest accuracy [6, 7]. Note that it is
possible to include several internal control proteins with distinct
molecular weights in the same sample. This provides the user with a
“plan B,” highly useful when the peak intensity of the regular
internal control is too weak or too intense as compared to the
peak of the protein of interest (hereafter referred to as the “PDZ
peak”), or when the regular internal control peak, but not the rest
of the electropherogram, has been altered due to a migration
defect. If any of the steps fails, the user can try to restart from
point 2 in the “Transforming the input data” section by varying the
default parameters.
3.1 Input Data
Extraction from the
Quantitative Capillary
Electrophoresis
Instrument
1. For each plate analyzed by holdup assay, open the software of
the electrophoresis instrument and make sure that all the ladders and samples are well aligned (see Note 1).
2. Give the proper names to the samples and export the electropherogram’s raw data including molecular weights (see
Note 2).
A Computational Protocol to Analyze PDZ/PBM Affinity Data Obtained. . .
65
2.1 Capillary
Electrophoresis
Instrument’s Software
Every capillary electrophoresis instrument has its specific software
to retrieve the data.
2.2 Computer
The use of a programming language is necessary to process all the
data. For this project, Python has been chosen.
2.3 SPIKE
Package and Software
Availability
A Python package is available in https://spikedoc.bitbucket.io
[12, 13]. The software can be found in free access here: https://
github.com/lio3867/BiDop
3 Methods
All mentioned steps were performed with in-house Python scripts
using different packages, including SPIKE. This protocol is meant
to be applied to both electropherograms recorded for the reference
and the sample. The protein extract loaded on the reference and the
sample resins should come from the same expression source and in
practice from the same storage tube. This protocol also requires an
internal control added directly to the common storage tube, before
starting the experiment. This control is usually a soluble protein
with an adequate molecular weight, avoiding in particular any
overlap with the molecular weight of the protein of interest. The
peak intensity of the internal control protein (hereafter referred to
as the “internal control peak”) is used for normalization so that the
two electropherograms obtained from the same source tube can be
directly compared with the highest accuracy [6, 7]. Note that it is
possible to include several internal control proteins with distinct
molecular weights in the same sample. This provides the user with a
“plan B,” highly useful when the peak intensity of the regular
internal control is too weak or too intense as compared to the
peak of the protein of interest (hereafter referred to as the “PDZ
peak”), or when the regular internal control peak, but not the rest
of the electropherogram, has been altered due to a migration
defect. If any of the steps fails, the user can try to restart from
point 2 in the “Transforming the input data” section by varying the
default parameters.
3.1 Input Data
Extraction from the
Quantitative Capillary
Electrophoresis
Instrument
1. For each plate analyzed by holdup assay, open the software of
the electrophoresis instrument and make sure that all the ladders and samples are well aligned (see Note 1).
2. Give the proper names to the samples and export the electropherogram’s raw data including molecular weights (see
Note 2).
A Computational Protocol to Analyze PDZ/PBM Affinity Data Obtained. . .
65