3. Fill the chambers with running buffer.
4. Load samples and ladders in the appropriate wells.
5. Run the gel following the manufacturer’s instructions (see
Note 8).
6. After electrophoresis, remove the gel from the tank and transfer
directly into the InstantBlue staining solution. Be sure that the
gel moves freely in stain to facilitate diffusion. Typically, about
20 ml is needed to cover the gel.
7. Colored proteins bands will start to develop immediately and a
suitable intensity is typically achieved after 15 min incubation at
room temperature with gentle shaking.
The protein sample is considered pure when no other band
other than the expected one for your protein of interest is detected
by SDS-PAGE using sensitive staining [20].
In the case of MAST2-PDZ, the protein is purified as previously described [36]. The 2-step purification process improved
drastically the purity of the sample as assessed by SDS-PAGE in
Fig. 7b. The GSTrap column allowed to remove a high content of
contaminants and to concentrate the MAST2-PDZ construct. The
SDS-PAGE gel of the eluted fractions showed a major band of the
protein and only a few contaminants extra-bands (Fig. 7b left
panel). Fractions containing MAST2-PDZ are pooled and then
purified by size-exclusion chromatography and the only band
remaining on the corresponding gel SDS-PAGE is the MAST2PDZ construct. A very weak band around 30 kDa slightly overlap
with the first fractions of MAST2-PDZ. The pool of fractions of gel
filtration is then concentrated and the gel SDS-PAGE revealed one
unique band indicative of the high degree of purity by SDS-PAGE
technique. This construct has a migration that does not match with
its calculated molecular weight as already observed. This might be
due to the effect of SDS binding [37] and/or the hydrophobicity
of the construct [38]. The limits of this technique are that the
detection is restricted to protein contaminants, and the detection
is proportional to the sample loading and gel staining and should be
confirmed by mass spectroscopy.
3.1.2 Capillary Gel
Electrophoresis (CGE)
Capillary gel electrophoresis (CGE) separates proteins according to
their molecular mass similarly to SDS-PAGE, and this methodology has been previously exploited to validate bacterial expression of
a library of 266 known human PDZ constructs developed for the
high-throughput “holdup” chromatographic assay for the determination of PDZ-PBM affinities [39]. Below we outline the main
steps of this high-throughput protocol.
1. Dilute to 1:8 (v:v), 1:16 (v:v), and 1:32 (v:v) each PDZ supernatant to be quantified.
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Ce ´ lia Caillet-Saguy et al.
4. Load samples and ladders in the appropriate wells.
5. Run the gel following the manufacturer’s instructions (see
Note 8).
6. After electrophoresis, remove the gel from the tank and transfer
directly into the InstantBlue staining solution. Be sure that the
gel moves freely in stain to facilitate diffusion. Typically, about
20 ml is needed to cover the gel.
7. Colored proteins bands will start to develop immediately and a
suitable intensity is typically achieved after 15 min incubation at
room temperature with gentle shaking.
The protein sample is considered pure when no other band
other than the expected one for your protein of interest is detected
by SDS-PAGE using sensitive staining [20].
In the case of MAST2-PDZ, the protein is purified as previously described [36]. The 2-step purification process improved
drastically the purity of the sample as assessed by SDS-PAGE in
Fig. 7b. The GSTrap column allowed to remove a high content of
contaminants and to concentrate the MAST2-PDZ construct. The
SDS-PAGE gel of the eluted fractions showed a major band of the
protein and only a few contaminants extra-bands (Fig. 7b left
panel). Fractions containing MAST2-PDZ are pooled and then
purified by size-exclusion chromatography and the only band
remaining on the corresponding gel SDS-PAGE is the MAST2PDZ construct. A very weak band around 30 kDa slightly overlap
with the first fractions of MAST2-PDZ. The pool of fractions of gel
filtration is then concentrated and the gel SDS-PAGE revealed one
unique band indicative of the high degree of purity by SDS-PAGE
technique. This construct has a migration that does not match with
its calculated molecular weight as already observed. This might be
due to the effect of SDS binding [37] and/or the hydrophobicity
of the construct [38]. The limits of this technique are that the
detection is restricted to protein contaminants, and the detection
is proportional to the sample loading and gel staining and should be
confirmed by mass spectroscopy.
3.1.2 Capillary Gel
Electrophoresis (CGE)
Capillary gel electrophoresis (CGE) separates proteins according to
their molecular mass similarly to SDS-PAGE, and this methodology has been previously exploited to validate bacterial expression of
a library of 266 known human PDZ constructs developed for the
high-throughput “holdup” chromatographic assay for the determination of PDZ-PBM affinities [39]. Below we outline the main
steps of this high-throughput protocol.
1. Dilute to 1:8 (v:v), 1:16 (v:v), and 1:32 (v:v) each PDZ supernatant to be quantified.
106
Ce ´ lia Caillet-Saguy et al.