2 Materials
2.1 Purity
2.1.1 Basic Reagents
for SDS-PAGE
Electrophoresis
1. Electrophoresis tank.
2. Power supply.
3. Polyacrylamide gels. Novex™ 4–12% Tris-Glycine Mini Gels
offers a broad range of molecular weight resolution from 6 to
400 kDa. Standard 10–12% polyacrylamide gels can also be
used to resolve PDZ domains.
4. 2Â Sample buffer: 100 mM Tris–HCl pH 6.8, 0.2% Bromophenol Blue (BBP), 4% SDS, 20% glycerol, 200 mM dithiothreitol (DTT) (see Note 2). Add 1 ml of 1 M Tris pH 6.8, a
pinch of BromoPhenol Blue (BPB) and vortex, 4 ml of 10%
SDS, 2 ml of 100% glycerol, and 2 ml of 1 M DTT.
5. Molecular-weight markers such as PageRuler™ Prestained
Protein Ladders from 10 to 180 kDa.
6. Migration buffer such as NuPAGE MES SDS Running Buffer.
Add 50 ml of the NuPAGE™ MES SDS Running Buffer (20Â)
and complete to 1 l with pyrolysis water.
7. Home-made or commercial protein staining solution. InstantBlue is a ready to use Coomassie protein stain for
polyacrylamide gels.
2.1.2 Capillary
Electrophoresis (CE)
1. Capillary electrophoresis system in this case a LabChip GX II
(PerkinElmer).
2. LabChip HT Protein Express Chip (PerkinElmer, 760499).
3. Protein Express Assay Reagent Kit (PerkinElmer,
CLS960008).
4. HT Protein 200 Sample Buffer (PerkinElmer 760518).
5. 384-well PCR plate (Greiner Bio-One) (see Note 3)
2.1.3 UV Spectroscopy
1. UV-Vis spectrometer.
2. Quartz cuvette suitable for UV (190–400 nm).
3. Sample buffer as described in Subheading 2.2, item 4.
4. Special wipe such as Kimwipes to clean the cuvette before
measuring.
2.2 Integrity
Measurements by
MALDI-TOF
2.2.1 Total Mass
1. MALDI steel plate.
A freshly prepared matrix solution: 25 mg/ml α-cyano-4hydroxycinnamic acid (HCCA) in 50% (v/v) acetonitrile,
0.1% (v/v) trifluoroacetic acid (TFA) in HPLC water.
2. ZipTip with C4 resin (Merck Millipore).
3. High quality protein standard from Bruker, Laserbio Labs, or
Sciex.
PDZ Sample Quality Assessment
103
2.1 Purity
2.1.1 Basic Reagents
for SDS-PAGE
Electrophoresis
1. Electrophoresis tank.
2. Power supply.
3. Polyacrylamide gels. Novex™ 4–12% Tris-Glycine Mini Gels
offers a broad range of molecular weight resolution from 6 to
400 kDa. Standard 10–12% polyacrylamide gels can also be
used to resolve PDZ domains.
4. 2Â Sample buffer: 100 mM Tris–HCl pH 6.8, 0.2% Bromophenol Blue (BBP), 4% SDS, 20% glycerol, 200 mM dithiothreitol (DTT) (see Note 2). Add 1 ml of 1 M Tris pH 6.8, a
pinch of BromoPhenol Blue (BPB) and vortex, 4 ml of 10%
SDS, 2 ml of 100% glycerol, and 2 ml of 1 M DTT.
5. Molecular-weight markers such as PageRuler™ Prestained
Protein Ladders from 10 to 180 kDa.
6. Migration buffer such as NuPAGE MES SDS Running Buffer.
Add 50 ml of the NuPAGE™ MES SDS Running Buffer (20Â)
and complete to 1 l with pyrolysis water.
7. Home-made or commercial protein staining solution. InstantBlue is a ready to use Coomassie protein stain for
polyacrylamide gels.
2.1.2 Capillary
Electrophoresis (CE)
1. Capillary electrophoresis system in this case a LabChip GX II
(PerkinElmer).
2. LabChip HT Protein Express Chip (PerkinElmer, 760499).
3. Protein Express Assay Reagent Kit (PerkinElmer,
CLS960008).
4. HT Protein 200 Sample Buffer (PerkinElmer 760518).
5. 384-well PCR plate (Greiner Bio-One) (see Note 3)
2.1.3 UV Spectroscopy
1. UV-Vis spectrometer.
2. Quartz cuvette suitable for UV (190–400 nm).
3. Sample buffer as described in Subheading 2.2, item 4.
4. Special wipe such as Kimwipes to clean the cuvette before
measuring.
2.2 Integrity
Measurements by
MALDI-TOF
2.2.1 Total Mass
1. MALDI steel plate.
A freshly prepared matrix solution: 25 mg/ml α-cyano-4hydroxycinnamic acid (HCCA) in 50% (v/v) acetonitrile,
0.1% (v/v) trifluoroacetic acid (TFA) in HPLC water.
2. ZipTip with C4 resin (Merck Millipore).
3. High quality protein standard from Bruker, Laserbio Labs, or
Sciex.
PDZ Sample Quality Assessment
103