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TitleCapillary Electrophoresis of Proteins and Peptides
Author
LanguageEnglish
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Total Pages345
Document Text Contents
Page 1

Edited by

Mark A. Strege
Avinash L. Lagu

Capillary
Electrophoresis

of Proteins
and Peptides

Volume 276

METHODS IN MOLECULAR BIOLOGYTMMETHODS IN MOLECULAR BIOLOGYTM

Edited by

Mark A. Strege
Avinash L. Lagu

Capillary
Electrophoresis

of Proteins
and Peptides

Page 2

Capillary Electrophoresis of Proteins and Peptides

Page 172

R
ecep

to
r–Ligan

d
In

teractio
n

s
1

5
9

Fig. 1. (A) Schematic of a standard ACE experiment. (B) Schematic of a partial-filling ACE experiment. (C) Schematic of a
flowthrough partial-filling affinity capillary electrophoresis experiment. Used with permission from ref. 3. (D) Schematic of a
multiple-step ligand-injection ACE experiment. Used with permission from ref. 4.

Page 173

160 Azad et al.

Fig. 2. (A) A representative series of electropherograms of Rist A in 0.192 M gly-
cine-0.025 M Tris-HCl buffer, pH 8.3, containing various concentrations of 1 using
the standard ACE technique. MO and CAB were used as internal standards. The total
analysis time was 2.0 min at 25 kV (current: 7.7 µA) using a 30.5-cm (inlet to detec-
tor), 50-µm id open, uncoated quartz capillary. (B) Scatchard plot of the data for Rist
A according to Eq 2. Used with permission from ref. 1.

are the measured migration times of the reference peak MO and Van, respec-
tively. A Scatchard plot can be obtained using Eq. 4. ∆MR,L is the magnitude of
the change in the mobility ratio (M) as a function of the concentration of 2. Eq. 4
allows for the estimation of Kb on a relative time scale and compensates for fluc-
tuations in voltage and/or capillary length.

10. Upon increasing the concentration of 2 in the capillary column a shift in the
migration time of Van is observed. The Van-2 complex is more negative than
Van and upon binding shifts to the right (longer migration time). The neutral

Page 344

Index 331

denaturation studies of protein charge
and size, 200, 201, 214

electrostatic interaction analysis,
affinity capillary electrophoresis,

204, 205, 207
carbonic anhydrase ligand binding,

207, 209
overview, 201, 203, 204
protein unfolding energetics, 209–213

hydrodynamic radius determination,
195–198, 200

net charge determination, 195–199, 213,
214

protein property measurement
advantages, 190, 213

synthesis,
carboxyl group amidation, 193, 214
lysine acetylation, 190, 192, 193, 214
materials, 191, 192, 213
overview, 190, 191, 213

Proteomics, see also Capillary isoelectric
focusing-mass spectrometry;
Microfluidics devices-mass
spectrometry; 2D gel
electrophoresis,

definition, 305, 306

QC, see Quality control
Quality control (QC), capillary

electrophoresis for protein
purity,

capillary electrophoresis-SDS method,
accuracy, 129, 130
heating of samples, 127, 135
linearity, 129
optimization, 126, 127
precision, 130, 132
robustness/ruggedness, 133
running conditions, 129, 135
sample preparation, 126
sensitivity, 130
specificity, 134
system suitability, 135

materials, 123
overview, 121, 122
SDS-polyacrylamide gel electrophoresis

accuracy and precision, 123, 124
Quenching, laser-induced fluorescence, 41–43

Rapid capillary gel electrophoresis,
capillary zone electrophoresis, 107–109
materials, 106
principles, 104, 105
recombinant formate dehydrogenase

expression monitoring in
Escherichia coli ,

culture time-dependence of
expression, 109–112

denaturing polyacrylamide gel
electrophoresis comparison,
110, 112

materials, 113
overview, 109
purification table, 114
separations, 115, 116

Recombinant proteins, see Formate
dehydrogenase; Insulin,
recombinant

Ristocetin A, affinity capillary
electrophoresis of binding to
D-Ala-D-Ala terminus
peptides, 156, 157, 166

Silane coatings,
acrylamide layers and substitutes, 16, 17
chemistry overview, 16, 18, 19
electro-osmotic flow measurements, 23–

25
fluorescent labeling of proteins and

peptides, 21, 23–25
instrumentation, 19–21
laser-induced fluorescence detection, 21,

24, 25
materials, 20, 21, 24
Si-C bond formation,

chlorination of silanol groups, 22–25
Grignard reaction, 23, 25
polymerization, 23, 25
pretreatment, 22

Si-O bond formation,
polymerization, 22, 25
pretreatment, 22
silane reaction, 22

steps, 16
Single cell protein fingerprinting,

instrumentation, 34, 36
materials, 34–36

Page 345

332 Index

on-column labeling and laser-induced
fluorescence detection,

chemistry, 32–34
extracted protein labeling and

electrophoresis, 35, 36
labeling reaction and electrophoresis,

34–36
single cell labeling, 35

protein extraction, 35
sheath-flow cuvet, 30–32

Theoretical plates, capillary electrophoresis,
15

2D gel electrophoresis,
microfluidics devices-mass spectrometry

analysis of bands, see
Microfluidics devices-mass
spectrometry

nano-scale liquid chromatography-mass
spectrometry, 306

principles, 189, 190, 306
proteomics, 306

Vancomycin, binding to D-Ala-D-Ala
terminus peptides,

multiple-step ligand injection affinity
capillary electrophoresis, 163,
164, 66

partial-filling affinity capillary electro-
phoresis, 157, 160, 161, 166

Warfarin-human serum albumin
interactions, see Human serum
albumin

Whole-column imaging detection,
axially illuminated fluorescence

detection, 242, 244
capillary zone electrophoresis studies of

protein binding, 246, 247
miniaturization, 244
overview, 230
protein detection in microfluidic devices,

239–241
protein-protein interactions, 236, 239
2D separation techniques, 241, 242

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