Applications of cell-free expressed protein microarrays

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Applications of cell-free expressed protein
microarrays
Applications of cell-free expression-based
protein microarrays have seen a rampant increase
over the last decade due to the several
advantages offered by these techniques over
traditional cell-based technologies.

• Harini Chandra
• Affiliations

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Master Layout (Application 1)
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This animation consists of 3 parts Application 1
- Biomarker detection Application 2 -
Immunological studies Application 3 - Protein
interaction studies
Detection of p53 autoantibodies in human serum
using cell-free expression based NAPPA
microarrays.
p53 positive sera
Anti-human IgG
Transcription translation
Patient sera
2
Cell-free lysate
p53 autoantibodies
cDNA
Anti-GST antibodies
3
BSA, BS3 printing mix
4
Protein microarray
5
Anderson, K. A., Ramachandran, N., Wong, J.,
Raphael,J. V. et al., Application of protein
microarrays for multiplexed detection of
antibodies to tumor antigens in breast cancer. J.
Proteome Res. 2008, 7, 14901499.
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Definitions of the componentsApplication 1
biomarker detection
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1. cDNA The complementary DNA expressing the
antigen of interest along with the GST tag. This
cDNA is transcribed and translated using suitable
cell-free lysate. The expressed protein is then
captured onto the microarray through the anti-GST
antibody. 2. BSA, BS3 printing mix The printing
mix for generation of NAPPA protein microarrays
consisted of bovine serum albumin (BSA), which
significantly increased the binding efficiency of
DNA, and BS3, which served as a protein
cross-linker. 3. p53 autoantibodies Antibodies
against the tumour suppressor gene, p53, is
believed to be one of the factors involved in
breast cancer. Early detection of such biomarkers
can have tremendous impact on disease treatment
and monitoring prognosis. An increasing number of
such biomarkers are being studied in order to
facilitate the early identification of
diseases. 4. Patient sera Suitable patient sera
samples were collected for the study. Sera was
obtained from breast cancer patients, early stage
ovarian cancer patients and healthy subjects. 5.
Anti-human IgG The binding of p53 autoantibody
to the corresponding antigen is detected by means
of anti-human IgG binding to the p53
autoantibody.
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Definitions of the componentsApplication 1
biomarker detection
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6. Protein microarray The microscope slide on to
which the DNA templates are immobilized and the
entire expression is carried out. The target
proteins bind to this array surface through an
antibody. 7. p53 positive sera Serum from those
patients who have autoantibodies to p53 is termed
as p53 positive. These patients will give
positive detection signals when probed by the
anti-human IgG.
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Application 1, Step 1
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Anti-human IgG
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Transcription translation
Patient sera
Cell-free lysate
p53 autoantibodies
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cDNA
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Action
Audio Narration
Description of the action
First show the base with the circular strands and
V-shaped objects bound to it. Then show the green
cloud appearing followed by the arrow and the
light purple objects. These must bind to the
V-shaped objects. Then show the yellow cloud with
maroon spade-shaped object. These must bind to
the purple object followed by binding of green
spade to the maroon spade.
The authors generated protein microarrays based
on NAPPA expression, which they probed with
diluted sera of breast cancer patients having p53
autoantibodies. Detection was carried out by
means of HRP-linked anti-human IgG.
The light purple shapes must appear and bind to
the blue V shaped objects.
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Application 1, Step 2
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p53 positive sera
p53 negative sera
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3
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Action
Audio Narration
Description of the action
First show the p53 negative sera followed by
the p53 positive sera with the red spots.
This study detected p53 autoantibodies by means
of NAPPA microarrays, which was confirmed by
ELISA. The p53 levels were found to be directly
related to tumour burden with serum antibody
concentration decreasing after neoadjuvant
chemotherapy.
The two rectangles must be shown as above.
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Master Layout (Application 2)
1
This animation consists of 3 parts Application 1
- Biomarker detection Application 2 -
Immunological studies Application 3 - Protein
interaction studies
Use of cell-free expression based protein
microarrays for detection of potential
immunogenic proteins of Plasmodium falciparum.
Proteins expressed
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E. coli IVTT
Spotting
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PCR amplified expression vectors
Reaction tube
Patient sera
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Naturally exposed group
Experimentally exposed group
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Doolan, D. L., Mu, Y., Unal, B., Sundaresh, S. et
al., Profiling humoral immune responses to P.
falciparum infection with protein microarrays.
Proteomics 2008, 8, 46804694.
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Definitions of the componentsApplication 2
immunological studies
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1. Reaction tube Cell-free expression of the
Plasmodium falciparum (Pf) proteins was carried
out in this reaction tube. 2. PCR amplified
expression vectors Vectors encoding 250 putative
Pf proteins were generated by PCR/recombination
cloning. These were expressed using the E. coli
IVTT. 3. E. coli IVTT Proteins were expressed
from the expression vectors using the E. coli
cell-free in vitro transcription-translation
(IVTT) mixture. The main drawbacks of this
bacterial expression system include the absence
of protein glycosylation and the lack of a proper
redox environment to allow for correct protein
folding and formation of disulphide bonds. 4.
Proteins expressed The 250 proteins were
individually expressed with greater than 90
efficiency and printed without any further
purification onto microarray slides.
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Definitions of the componentsApplication 2
immunological studies
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5. Patient sera The printed microarray slides
were then probed with serum samples from
different patient groups a) Naturally exposed
group Group of (Kenyan) subjects that had been
naturally exposed to Pf and had reported clinical
symptoms within the previous year. b)
Experimentally exposed group Volunteers who had
never been infected were immunized with radiation
attenuated Pf and then challenged with the bites
of 5 infected mosquitoes. Samples were collected
from these subjects prior to immunization,
immediately prior to challenge and following
challenge.
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Application 2, Step 1
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Protein expression and array printing
Proteins expressed
E. coli IVTT
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PCR amplified expression vectors
Array printing
Reaction tube
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Action
Audio Narration
Description of the action
(Please redraw all images.) First show the grey
solution in the tube which must be zoomed into.
The figures on the right must then be shown
followed by the curved arrow and then the
coloured shapes. The figure below must then
appear and the black shape must be shown to move
horizontally from left to right.
The authors carried out cell-free expression of
the PCR amplified vectors using an E. coli IVTT.
They expressed 250 putative proteins that were
printed directly onto microscopic array slides
without any purification.
The grey solution must be zoomed into and the
figures on the right must be shown.
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Application 2, Step 2
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Array probing and analysis
Experimentally exposed group
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Pre-immunization
Anti-polyhistidine mouse Ab
Patient sera
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Naturally exposed group
Post-challenge
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Action
Audio Narration
Description of the action
These arrays were probed with serum samples from
patients who had been naturally exposed to Pf and
who were experimentally exposed by means of
radiation attenuated Pf. Authors successfully
identified 72 highly immunoreactive protein
antigens as well as 56 previously uncharacterized
antigens that were serodominant, which can serve
as potential vaccine targets.
First show the four black parallelograms with
their labels followed by appearance of the cloud
shaped figures on top of them. This is then
followed by appearance of the black rectangles
with coloured dots.
The cloud figures must appear on the black
parallelograms followed by the black rectangles
with coloured spots.
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Master Layout (Application 3)
1
This animation consists of 3 parts Application 1
- Biomarker detection Application 2 -
Immunological studies Application 3 - Protein
interaction studies
Identification of novel protein-protein
interactions using NAPPA microarray.
Cell-free lysate
Expressed proteins
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Anti-GST antibodies
3
Replication initiation DNAs
Query proteins
4
Protein microarray
Protein interaction studies
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Ramachandran, N., Hainsworth, E., Bhullar, B.,
Eisenstein, S. et al., Self-assembling protein
mircoarrays. Science 2004, 305, 8690.
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Definitions of the componentsApplication 3
Protein interaction studies
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1. Replication initiation DNAs Human genes for
29 different proteins involved in the initiation
of DNA replication were immobilized on to the
array surface and expressed in duplicate using
NAPPA. 2. Cell-free lysate Rabbit reticulocyte
lysate was applied as a single continuous layer
across the slide and used for expression of the
immobilized DNA. 3. Expressed proteins The
replication initiation proteins were expressed on
the array surface where they became immobilized
via the anti-GST antibodies. Signals were
detected readily and were found to show high
reproducibility between duplicates. 4. Protein
interaction studies Protein interaction studies
were carried out between each of the 29 proteins
expressed and a duplicate array of the same 29
proteins used as a query molecule, thereby
generating a 29 X 29 interaction matrix. Such
interaction studies can also be carried out with
other protein or peptide molecules, as well as
other biomolecules. These provide insights into
pathway mapping, protein function determination
and substrate identification. 5. Query protein
The protein molecule that is used to probe the
protein array of interest in order to detect any
possible interaction is known as the query
protein.
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Application 3, Step 1
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Protein expression using NAPPA
Expressed proteins
Cell-free lysate
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Anti-GST antibodies
Replication initiation DNAs
3
Protein microarray
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Action
Audio Narration
Description of the action
First show the gray array surface below followed
by one of the spots being zoomed into and
appearance of the figures shown above. Show the
base with the circular strands and green spade
shaped objects bound to it. Then show the violet
cloud appearing followed by the arrow and the
purple and blue objects. These must bind to the
spade-shaped objects.
Ramachandran et al., tested the use of NAPPA
microarrays by immobilizing 29 sequence-verified
human genes involved in replication initiation on
the array surface and then expressing them in
duplicate with RRL. The expressed proteins bound
to the anti-GST antibodies present on the array
surface.
One of the spots on the array below must be
zoomed into and the events drawn above must be
shown.
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Application 3, Step 2
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29 x 29 protein interaction matrix
Protein interaction studies
Query proteins
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3
Array with expressed immobilized target proteins
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Action
Audio Narration
Description of the action
First show the grey array surface with spots
followed by the binding of the coloured proteins
to this surface. Once these move about and
finally bind to the grey surface, the figure on
the right must appear.
The authors made use of each of these expressed
proteins to probe another duplicate array of the
same 29 proteins thereby generating a 29 x 29
protein interaction matrix. 110 interactions were
detected between proteins of the replication
initiation complex, of which 63 were previously
undetected ones.
The coloured query proteins must probe the grey
array surface below.
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Interactivity option 1Step No 1
1
Match the following applications to their
appropriate studies to understand more about them.
a) Identification of serodiagnostic tests and
vaccine development against Coxiella burnetti.
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1. Biomarker discovery
b) High density NAPPA array approach for studying
well characterized gene pairs
2. Immunological studies
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c) Rapid bead-based assay for multiplexed
detection of antibodies to EBNA-1 and p53.
3. Protein interaction studies
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Boundary/limits
Interacativity Type Options
Results
Correct answer is 1-c, 2-a and 3-b. Once the user
gets this correct match, he is redirected to
steps 2, 3 and 4 to view the animations shown
there.
User should be allowed to drag and drop the
options on the left onto the options in the
right. Once the correct match is made, user is
redirected to steps 2, 3 4.
Match the following drag and drop.
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Interactivity option 1Step No 2(a)
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Biomarker discovery rapid bead-based assay for
multiplexed detection of antibodies to EBNA-1 and
p53.
Luminex beads of different fluorescence
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Anti-GST Abs coupled to beads
Mixing
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Expressed protein captured onto beads
Antigen coated beads
Cell-free expression of GST-tagged DNA
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Action
Audio Narration
Description of the action
First show the three cans on left top with the
colored figures. Next show the cans below with
the colored strands. These cans must be mixed
together according to their color to give rise to
the three cans in the middle panel. Again these
three must be mixed to give the can on the right.
The authors developed a programmable multiplexed
immunoassay where tagged antigens were expressed
using in vitro transcription and translation and
captured onto anti-tag coated beads. The
synthesized proteins were immobilized on to the
beads through the capturing agent. These beads
were then mixed together.
As shown in animation.
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Wong, J., Sibani, S., Lokko, N. N., LaBaer, J.,
Anderson, K. S., Rapid detection of antibodies in
sera using multiplexed self-assembling bead
arrays. J. Immunol. Methods 2009, 350, 171182.
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Interactivity option 1Step No 2(b)
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Biomarker discovery rapid bead-based assay for
multiplexed detection of antibodies to EBNA-1 and
p53.
Substrate
IgG-containing human serum
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Colored product detected
Enzyme linked secondary Abs
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Action
Audio Narration
Description of the action
Show the yellow solution containing inverted Y
shaped objects moving into the can and binding to
the green shapes. Next, show the green inverted Y
which must bind to the black inverted Y. Next the
grey sun shaped object must appear which must
bind to the brown part of the Ab and once
binding occurs, it must turn bright green as
shown in animation.
Serum was added to these coupled beads and human
IgG detected by probing with enzyme-linked
anti-human IgG. Colored reaction was observed on
addition of substrate to the enzyme. The authors
demonstrated this approach for detection of
antibodies to Epstein-Barr virus nuclear antigen
1 (EBNA1) and p53.
As shown in animation.
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Wong, J., Sibani, S., Lokko, N. N., LaBaer, J.,
Anderson, K. S., Rapid detection of antibodies in
sera using multiplexed self-assembling bead
arrays. J. Immunol. Methods 2009, 350, 171182.
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Interactivity option 1Step No 3(a)
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Immunological studies identification of
immunogens of Q-fever-causing Coxiella burnetti.
Proteins expressed
E. coli IVTT
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Array printing with crude IVTT lysate
1988 PCR amplified ORFs
96 well reaction plate
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Nitrocellulose microarrays
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Action
Audio Narration
Description of the action
Beare et al., carried out in vitro transcription
and translation of 1988 ORFs of C. burnetti using
E. coli based cell-free systems. 75 of the ORFs
were successfully generated as full-length
proteins and spotted onto nitrocellulose arrays.
As shown in animation.
(Please redraw all images.) Show the black plate
on the left. One of the spots must be zoomed into
and the next figure must appear which must then
give rise to the proteins. These must be shown
to move onto the array surface and the robotic
arm must move across the surface.
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Beare, P. A., Chen, C., Bouman, T., Pablo, J. et
al., Candidate antigens for Q fever serodiagnosis
revealed by immunoscreening of a Coxiella
burnetii protein microarray. Clin. Vaccine
Immunol. 2008, 15, 17711779.
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Interactivity option 1Step No 3 (b)
1
Immunological studies identification of
immunogens of Q-fever-causing Coxiella burnetti.
Sera from vaccinated patients
Sera from acute Q-fever patients
2
75 of the ORFs generated full length proteins
successfully. 50 C. burnetti proteins were found
to react strongly with immune sera.
3
4
Action
Audio Narration
Description of the action
The arrays were probed with sera from patients
who had been vaccinated as well as acute Q-fever
patients. 50 proteins were identified that were
found to react strongly with the immune sera.
As shown in animation.
Show the grey parallelograms above which must
then be covered with two solutions of slightly
different color. Once this happens the array
signals shown below must be displayed.
5
Beare, P. A., Chen, C., Bouman, T., Pablo, J. et
al., Candidate antigens for Q fever serodiagnosis
revealed by immunoscreening of a Coxiella
burnetii protein microarray. Clin. Vaccine
Immunol. 2008, 15, 17711779.
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Interactivity option 1Step No 4 (a)
1
Protein interaction studies - high density NAPPA
array approach for studying well characterized
gene pairs
Expressed proteins
Cell-free lysate
Unbound query proteins
cDNA of query protein without GST tag
2
Anti-GST antibodies
647 unique genes printed on array
3
High density protein microarray
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Action
Audio Narration
Description of the action
The authors made use of high density NAPPA arrays
to study protein interactions. 647 unique genes
were printed on to the array surface and
expressed by adding the cell-free lysate. cDNA of
the query protein was also added to the same
mixture such that the query was co-expressed but
remained unbound due to the lack of a tag
capturing agent.
As shown in animation.
Show the grey surface with spots below. One spot
must be zoomed into and the surface above must be
show with the Y shaped objects and concentric
circles bound to it. Unbound concentric circles
must also appear along with the violet cloud.
This must be shown to give rise to proteins as
shown.
5
Ramachandran, N., Raphael, J. V., Hainsworth, E.,
Demirkan,G. et al., Next-generation high-density
self-assembling functional protein arrays. Nat.
Methods 2008, 5, 535538.
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Interactivity option 1Step No 4 (b)
1
Protein interaction studies - high density NAPPA
array approach for studying well characterized
gene pairs
Antibodies to detect protein interactions
2
Protein interactions using Jun, Fos and MDM2 as
queries detected
3
Protein interaction studies
Action
Audio Narration
Description of the action
4
The arrays were then probed with antibodies
specific to the query protein. Authors detected
various protein interactions using well known
query proteins.
Show colored proteins binding to the grey surface
followed by addition of the inverted Y shaped
antibodies which must bind to the colored
proteins. This binding must generate the signal
shown on the right.
As shown in animation.
5
Ramachandran, N., Raphael, J. V., Hainsworth, E.,
Demirkan,G. et al., Next-generation high-density
self-assembling functional protein arrays. Nat.
Methods 2008, 5, 535538.
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Questionnaire
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• 1. Which of the following provides improved DNA
  binding to the microarray during NAPPA? Answers
  a) BS3 b) GST c) BSA d)? Avidin
• 2. Which of these expression systems will not
  allow for protein glycosylation?
• Answers a) Rabbit Reticulocyte Lysate b) E.
  coli S30 c) Wheat germ extract d)? All of the
  above
• 3. How many immunoreactive protein antigens of P.
  falciparum were detected in the study by Doolan
  et al.?
• Answers a) 20 b) 45 c) 250 d)? 72
• 4. How many previously uncharacterized protein
  interactions were detected by Ramachandran et al.
  (2004) in their 29 x 29 interaction matrix?
• Answers a) 110 b) 56 c) 37 d)? 63
• 5. C. burnetti causes which of the following
  infections?
• Answers a) Breast cancer b) Ovarian cancer c)
  Malaria d)? Q-fever

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Links for further reading

• Books
• New and Emerging Proteomic Techniques. Edited by
  Dobrin Nedelkov Randall W.Nelson (Humana
  Press).
• Research papers
• Chandra, H. Srivastava, S. Cell-free
  synthesis-based protein microarrays and their
  applications. Proteomics 2010, 10, 1-14.
• Davies, D. H., Liang, X., Hernandez, J. E.,
  Randall, A. et al., Profiling the humoral immune
  response to infection by using proteome
  microarrays high-throughput vaccine
  anddiagnostic antigen discovery. Proc. Natl.
  Acad. Sci. USA 2005, 102, 547552.
• Eyles, J. E., Unal, B., Hartley, M. G., Newstead,
  S. L. et al., Immunodominant Francisella
  tularensis antigens identified using proteome
  microarray. Proteomics 2007, 7, 21722183.
• Lopez, J. E., Beare, P. A., Heinzen, R. A.,
  Norimine, J. et al., High-throughput
  identification of T-lymphocyte antigens from
  Anaplasma marginale expressed using in vitro
  transcription and translation. J. Immunol.
  Methods 2008, 332, 129141.
• Anderson, K. A., Ramachandran, N., Wong, J.,
  Raphael,J. V. et al., Application of protein
  microarrays for multiplexed detection of
  antibodies to tumor antigens in breast cancer. J.
  Proteome Res. 2008, 7, 14901499.

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Links for further reading

• Research papers
• Doolan, D. L., Mu, Y., Unal, B., Sundaresh, S. et
  al., Profiling humoral immune responses to P.
  falciparum infection with protein microarrays.
  Proteomics 2008, 8, 46804694.
• Ramachandran, N., Hainsworth, E., Bhullar, B.,
  Eisenstein, S. et al., Self-assembling protein
  mircoarrays. Science 2004,305, 8690.
• Wong, J., Sibani, S., Lokko, N. N., LaBaer, J.,
  Anderson, K. S., Rapid detection of antibodies in
  sera using multiplexed self-assembling bead
  arrays. J. Immunol. Methods 2009, 350, 171182.
• Beare, P. A., Chen, C., Bouman, T., Pablo, J. et
  al., Candidate antigens for Q fever serodiagnosis
  revealed by immunoscreening of a Coxiella
  burnetii protein microarray. Clin. Vaccine
  Immunol. 2008, 15, 17711779.
• Ramachandran, N., Raphael, J. V., Hainsworth, E.,
  Demirkan,G. et al., Next-generation high-density
  self-assembling functional protein arrays. Nat.
  Methods 2008, 5, 535538.