Protein microarray

Presentation by – Aditya Shandilya

 Introduction
• A protein microarray (or protein chip) is a
high-throughput method used to track the
interactions and activities of proteins .
• Determine their function .
• Determining function on a large scale.
• Its main advantage lies in the fact that large
numbers of proteins can be tracked in parallel
 Master Layout (Part 1)
1
This animation consists of 4 parts:
Part 1 – Need for protein microarrays
Part 2 – Protein expression & purification
Part 3 - Array functionalization & printing
Part 4 – Protein detection & analysis

2
Protein analysis

Protein sample

3 Protein analysis

Protein microarray

4
Several protein samples

5
1 Definitions of the components:
Part 1 – Need for protein microarrays

1. Protein microarray: These are miniaturized arrays normally made of

2 glass, polyacrylamide gel pads or microwells, onto which small quantities
of many proteins are simultaneously immobilized.
2. Protein analysis: Proteins can be studied for their structural and
functional aspects depending upon the requirement. Analysis
procedures, until recently, involved long and tedious protocols that
3 proved to be too difficult with an increasing number of protein samples.
The analysis process was facilitated significantly with advent of protein
microarrays.

5
1 Part 1, Step 1:

2
Protein sample

3
4
Action Description of the action Audio Narration
The clock must (Please redraw all figures.) Functional analysis of proteins is a time
be shown to tick First show the protein sample followed by the consuming process that requires many steps.
such that the clock on top. The arms on the clock must Analysis of a single protein at a time would be a
time changes move slowly as each arrow keeps appearing tedious and laborious process.
from 3 to 8 as followed by appearance of the green figure
5 shown. on the right and the clock above.
1 Part 1, Step 2:

3
Protein microarray

Protein microarrays allow analysis

of thousands of samples
simultaneously!

4
Action Description of the action Audio Narration
Multiple protein (Please redraw all figures.) Analysis of several protein samples will
samples must be First show several protein tubes appearing undoubtedly take a long time if they are run
shown to appear one in sequence as shown in the animation one at a time. Protein microarrays successfully
at a time as shown followed by the hands in the clock above
followed by constant moving around continuously. Then the grey
overcame this hurdle by allowing analysis of

5 ticking of the clock

until it completes
array figure on right must appear and
hands of the clock must stop moving.
several samples simultaneously.

several circles.
 Master Layout (Part 2)
1
This animation consists of 4 parts:
Part 1 – Need for protein microarrays
Part 2 – Protein expression & purification
Part 3 - Array functionalization & printing Gene insert
Part 4 – Protein detection & analysis

Protein

2 Gene of interest
expression

Heterologous host (e.g.

Protein
Expression purification
vector

3
Chromatography

4
Purified protein Protein purity
of interest tested
SDS-PAGE

5
Biochemistry by A.L. Lehninger, 3 rd edition (ebook)
 Definitions of the components:
1 Part 2 – Protein expression & purification

1. Expression vector: An expression vector or construct is used to

introduce and express a particular gene of interest in a target cell. An
2 efficient expression vector system must be capable of producing large
quantities of the protein product.
2. Heterologous host: A system such as E. coli that will provide the
required cellular machinery for transcription and translation of the gene of
interest which is introduced by means of a suitable expression vector.
3 3. Gene of interest: The gene that codes for the desired target protein that
needs to be printed on the surface of the microarray.
4. Gene insert: The gene sequence that is taken up by the plasmid vector
for further expression is then referred to as an insert.
4 5. Protein expression: The host system carries out transcription and
translation using its cellular machinery to express the protein of interest
along with its own proteins as well.

5
 Definitions of the components:
1 Part 2 – Protein expression & purification

1. Expression vector: An expression vector or construct is used to

introduce and express a particular gene of interest in a target cell. An
2 efficient expression vector system must be capable of producing large
quantities of the protein product.
2. Heterologous host: A system such as E. coli that will provide the
required cellular machinery for transcription and translation of the gene of
interest which is introduced by means of a suitable expression vector.
3 3. Gene of interest: The gene that codes for the desired target protein that
needs to be printed on the surface of the microarray.
4. Gene insert: The gene sequence that is taken up by the plasmid vector
for further expression is then referred to as an insert.
4 5. Protein expression: The host system carries out transcription and
translation using its cellular machinery to express the protein of interest
along with its own proteins as well.

5
1 Definitions of the components:
Part 2 – Protein expression & purification
6. Protein purification: Since the target protein is expressed along with
other proteins native to the host system, it is essential to purify the
2 desired protein prior to printing on to the array surface. For this reason,
the gene of interest is often fused with a convenient tag sequence such
as His6 that will facilitate the purification process.
7. Chromatography: A convenient purification technique that separates
proteins based on properties such as size, net charge or specific affinity
3 towards a particular molecule or ligand. Several advancements have been
made in chromatographic techniques that allow for accurate protein
separation.
8. 2-D electrophoresis: This is another protein separation technique
which is more commonly used for finer protein separation. It is a
4 combination of isoelectric focusing in the first direction and SDS-PAGE in
the second direction, thereby bringing about protein separation based on
their isoelectric points (net charge) as well as their molecular weights.
9. Purified protein of interest: Once the purification is complete, the
desired protein of interest is obtained which can then be used for
5 spotting onto the array surface.
1 Part 2, Step 1:
Gene insert
Protein expression
machinery

2 Heterologous host
Gene of interest

Expression
vector

3 Transcription Translation

mRNA
Expressed proteins

4
Action Description of the action Audio Narration
The green arc must First show the figure on the top left with the The gene coding for the protein of interest is
be incorporated in multicolor circle. Next, show the green arc expressed in a suitable heterologous host system
the multicolor circle. being incorporated as part of this circle
The second figure followed by appearance of the colored circles
such as E. coli by means of expression vectors like
must then be plasmids. The host cell machinery is used for
and brown figures. This must be zoomed into
5 zoomed into and
animation below
and the animation sequence shown below
must be shown.
transcription and translation which results in a
mixture of proteins consisting of the target protein
shown. along with other host proteins.
 Part 2, Step 2:
1 His6 tagged
protein Purified
protein
Protein of
interest

2 Direction of
Ni-NTA
Separation
based on
coated MW
migration beads

3
Chromatographic SDS-PAGE
purification

Protein of interest

4
Unwanted proteins

Action Description of the action Audio Narration

As shown (Please redraw all figures.) Since the protein of interest is expressed along with
in the First show the mixture in the can on top along with the first column. Then show the other proteins native to the host, it is essential to purify
animation. mixture in the can being poured into the column followed by appearance of the flask on the target protein before it can be used for microarray
top. The red and blue objects must then come out of the column into the tube below. applications. This can be done by chromatographic
Next the second column and tube must appear and from this the yellow and purple procedures to obtain the pure target protein. Protein
purity is tested on SDS-PAGE gels. Tags like His6 are

5
objects must come down into the tube below and the ‘unwanted proteins’ label must
appear. Finally the third column and tube must appear and the green object must come often fused with the protein of interest to facilitate the
into the tube. The figure on the right must be shown with the 3 tubes and the blue slab. purification process due to its specific affinity towards
The arrow marks with labels must appear followed by colored spots. The green spot and nickel.
tube must then be highlighted as shown.
Biochemistry by A.L. Lehninger, 3 rd edition (ebook)
 Master Layout (Part 3)
1
This animation consists of 4 parts:
Part 1 – Need for protein microarrays
Part 2 – Protein expression & purification NH2 NH2
Part 3 - Array functionalization & printing Si Si
Si
Part 4 – Protein detection & analysis NH2 NH2
NH2 CHO CHO CHO O OO O
CHO O O
CHO

2 Array functionalization

Si Si
Si
CHO CHO CHO O OO O
CHO CHO O O

Protein microarray
Robotic array printing

5
 Definitions of the components:
1 Part 3 – Array functionalization & printing

1. Array functionalization : The microarray surface must be suitably

derivatized with a chemical reagent that can react with the groups present
2 on the protein surface in order to firmly immobilize them on the
microarray. Functionalization is often done with silane derivatives as these
react easily with the groups present on the protein. Aldehyde groups react
with amine groups present on the protein to form Schiff’s base linkages
which hold the protein firmly in place.

3 2. Robotic array printing: High-precision contact-printing robots are

commonly used to spot nanoliter or picoliter quantities of protein solution
onto the array surface. The proteins are usually mixed with glycerol
solution in order to prevent them from evaporating.

5
1 Part 3, Step 1:
Commonly used array surfaces
Glass Gold Nitrocellulose Hydrogel

2
Reactivity Moderate Low Moderate Moderate

3
Applicability No Yes No No
for mass
spectrometry

Surface Low Low High High

absorption

4
Action Description of the action Audio Narration
First the Commonly used array surfaces include glass, gold, nitrocellulose and
First show the parallelogram figures
figures on hydrogels. While glass slides are easy to handle, available at low cost
on top with their respective labels
top must and can be used with existing scanning equipment, they show relatively
followed by the table below, one row
appear low surface absorption and need to be derivatized with more reactive
at a time.
followed by groups. They however continue to be used more extensively than the
5 the table
below.
other array surfaces, which are significantly more expensive.
Comparative features for commonly used array surfaces are
demonstrated in this table.
1 Part 3, Step 2:
Array functionalization

Si Si
Si
CHO CHO CHO O OO O
CHO CHO O O

3
Protein microarray

4
Action Description of the action Audio Narration
The hand must (Please redraw all figures.) The array surface is functionalized with a suitable
move as shown in First show the parallelogram with spots. Then chemical reagent that will react with groups
animation and red show the hand moving from left to right
cloud must appear. present on the protein surface. Aldehyde and silane
along the spots followed by appearance of
The spot must then the red cloud and disappearance of the hand. derivatizations are commonly used as they interact

5 be zoomed into and

the figure above
must appear.
The spots are then zoomed into and the
structures above must be shown.
well with amino groups present on the protein
surface resulting in firm capture of the protein.
 Part 3, Step 3:
1
Robotic array printing

Target protein

Derivatized array Robotic arm NH2 NH2

NH2 CHO CHO CHO
surface
2 CHO CHO
Aldehyde
functionalization

3 NH2
Si Si
O O O OO O
NH2
Si
Silane
derivatization

4
Action Description of the action Audio Narration
The black robotic The protein solution is printed on to the array surface in extremely
(Please redraw all figures.) small volumes by means of a robotic printing device that has small
arm must move First show the picture with the
across the pins attached to it for this purpose. The slides are kept for a suitable
black machine arm moving across duration following the printing step to allow capture of the protein
translucent the translucent surface from
surface after on to the array surface. The unreacted sites are then quenched by a
bottom to top. Then zoom into the
5 which the figure
must be zoomed
translucent surface to show the
figure on the right.
blocking solution such as BSA which also prevents any non-specific
protein binding in subsequent steps.
into.
 Part 3, Step 4:
1 Types of arrays
Forward phase Reverse phase
arrays arrays
Probe antibody
Test lysate

2 Specific antigen
binding Test lysate
Capture antibody immobilized
immobilized Direct labeling

3
4
Action Description of the action Audio Narration
The dots must be First show the parallelogram below There are two types of protein arrays that are commonly used. In
zoomed into and followed by zooming into the dots. Then forward phase arrays, the analyte of interest such as an antibody or
the two figures show the figure on left top with the blue aptamer is bound to the array surface and then probed by the test
above muse be circles binding to the V shaped objects lysate which may contain the antigen of interest. In reverse phase
shown as in the below. Similarly, then show the figure on arrays, however, the test cellular lysate is immobilized on the array
5 animation. right with the V shaped objects moving
down and binding to the blue circles.
surface and then probed using detection antibodies specific to the
target of interest.
 Master Layout (Part 4)
1
This animation consists of 4 parts:
Part 1 – Need for protein microarrays
Part 2 – Protein expression & purification
Part 3 - Array functionalization & printing
Part 4 – Protein detection & analysis

2 Labelled target proteins Array slide

Capture antibodies

Direct labeling Microarray scanner

3

Labelled secondary
antibody
4 Capture antibodies

Sandwich assay
Data analysis
5
 Definitions of the components:
1 Part 4 – Protein detection & analysis

1. Direct labelling : Here, all the target proteins are labelled with a
fluorescent or radioactive tag which allow direct detection upon binding to
2 the immobilized capture antibody on the array surface.
2. Capture antibodies: The antibodies (or sometimes other molecules like
aptamers) that have been immobilized on to the array surface for specific
capture of a particular analyte.

3 3. Labelled target proteins: The target proteins are directly labelled by

means of a fluorescent or radioactive tag which can be easily detected by
scanning techniques or radioactivity measurement after the protein gets
captured on to the array surface.
4. Sandwich assay: In this detection technique, a secondary antibody
4 recognizing a different epitope on the same target analyte is labelled with a
suitable tag and used for detection.
5. Labelled secondary antibody: An antibody that can recognize an epitope
different from the one recognized by the capture antibody can be used for
detection purposes. This secondary or detection antibody is labelled with a
5 convenient fluorescent dye which is viewed in the microarray scanner upon
antigen-antibody binding.
 Definitions of the components:
1 Part 4 – Protein detection & analysis

6. Microarray scanner: The microarray slide is scanned at wavelengths

suitable to detect the fluorescently tagged proteins. The output from this
2 scanner is generated on a connected computer device.
7. Data analysis: The protein array data can be analyzed by means of
several available software to understand which proteins have been printed
and subsequently detected.

5
1 Part 4, Step 1:
Detection techniques

Labelled secondary
Labelled target proteins antibody

2
Capture antibodies Capture antibodies

3 Direct labelling Sandwich assay

4
Action Description of the action Audio Narration
As shown in First show the figure on the left with the Y shaped In the direct labelling detection technique, all the target
animation. objects on the grey surface. Then show the blue proteins are labelled with a fluorescent or radioactive tag
teardrop binding to the Y shaped object with colour that facilitates easy detection upon binding to the
of the sun changing from grey to orange. Then immobilized capture antibody on the array surface. In the
show the figure on the right and the red inverted Y sandwich assay, however, a fluorescent tagged secondary

5 shaped object binding to the blue teardrop with

change in colour of the sun from grey to bright
antibody that recognizes a different epitope on the target
antigen binds to it and is detected by means of the
green. fluorescence.
 Part 4, Step 2:
1
Microarray scanner

3 Data analysis

4
Action Description of the action Audio Narration
The orange and (Please redraw all figures.)
The protein microarray is then scanned at in a microarray
green beams must First show the instrument on the left scanner that allows detection of the fluorescently labeled
be shown to top followed by the orange and green proteins or antibodies. The output from this scanner is
appear instrument beams coming out of it and the figures then received by an appropriate software on which the
shown on the left. placed below. Then show the arrow
5
data can be analyzed.
mark and appearance of the computer
screen on the right.
1 Interactivity option 1:Step No: 1
Certain well characterized proteins are printed on the array below with their corresponding
query molecules shown on the left labeled with differently fluorescing dyes. Match the protein
interacting pairs, Jun & Fos, p53 & MDM2, by dragging the query to the correct protein on the
array surface in order to see the signal output.

2 p53

Jun

3
MDM2 Signal output
Fos

4
Interacativity Type Options Boundary/limits Results
Once the user matches the green
Drag and drop. User must drag and
shape to the orange dots, its color
drop the figures

5
must change to blue and once the
given on the left to
purple shape is matched with pink
their corresponding
dots, its color must become red.
coloured circles on
Then the figure on the right must
the grey surface.
appear.