Product Overview
Proteome at Your Fingertips
Applications for the Ab Microarray 500
• Applications—biomarker discovery, cancer research, profiling disease states & more
• Rapid & flexible—Screen >500 antibodies in 1 day; analyze data in 1 hour
≥80% correlation with Western blot analysis
• Highly reliable—≥
• Validated technology—over 40 papers to date
Clontech’s Ab Microarray 500 is a robust, inexpensive tool for high-throughput analysis of proteomic profiles.
This array can be used as a screening tool for biomarker discovery and target identification—as well as for rapid
expression profiling of proteins in a variety of samples, including serum, tissues, cell lines, and cancer vs. normal
or treated vs. untreated samples. For example, this technology has been used to identify overexpressed proteins
in mantle cell lymphoma (1), yielding results that were validated using Western blot and immunohistochemistry.
The Technology
The Ab Microarray 500 is designed to perform rapid, reliable characterization of changes in protein expression between two samples.
It allows scientists to focus their research on a smaller number of proteins that have biological relevance—by screening >500 proteins
in one day. This array, which contains specific monoclonal antibodies, is incubated with Cy3™/Cy5™-labeled serum, tissue, or cell extract
samples—in order to profile disease states, identify tumor-associated antigens, perform time course studies, discover biomarkers,
and more. The antibodies on the array recognize human, mouse, and rat proteins.
High data quality is achieved by using a Cy3/Cy5 dye swap detection technology together with an internally normalized ratio (2).
This technology also has a built-in concentration step that allows for the detection of low-abundance proteins by accumulating antigens
onto the antibody during the incubation step—thus increasing the signal. A one-hour data analysis procedure yields highly sensitive,
reproducible, and reliable data that are biologically relevant. These data have been validated using Western blotting, ELISA, immunohistochemistry, and literature references (over 40 publications to date).
Microarray with >500 pairs
of unique monoclonal antibodies
Array incubated with
native protein extracts
Native antigens bound
to antibodies on array
(cells, tissues, body fluids, etc.)
ay 500
icroarr
Ab M
ay 500
icroarr
Ab M
Labeled analyte
Western blot validation
Capture antibody
Array surface
ELISA validation
Immunohistochemistry
validation
Literature references
Overview of the Ab Microarray 500 Protocol.
1 4 Clontech Laboratories, Inc. • www.clontech.com
Clontechniques April 2009
Normalized intensity (log scale)
Product Overview
A
10¹
Tonsil
MCL 1
MCL 2
MCL 3
CRIK
10 0
Hsp90
1
2
3
4
5
6
MCL samples
Control
MDM2
7 7 repeated
Figure 1. Expression levels of the 13 overexpressed proteins
in the MCL samples as compared to the control. The data points
are colored in red/orange, indicating overexpression. The samples
are numbered 1 to 7. MCL7 showed a reversed expression pattern
compared to the other 6 MCL samples. MCL7 was tested twice
and demonstrated a consistent expression pattern. Data were
analyzed using GeneSpring™ software (Agilent Technologies, Inc.)
B
Data Reproducibility
Six different histologically-confirmed MCL samples displayed the
same protein expression profile, demonstrating the reliability of the
array in providing consistent, accurate results. The reproducibility of
the array data was also confirmed by the MCL7 sample that was
analyzed twice (in 2 different experiments) and yielded the same result.
Validation of Results
The Ab Microarray 500 data from the mantle cell lymphoma
analysis (Figure 1) was validated by Western blot (1; data not
shown) and immunohistochemistry (Figure 2).
False Positives
Western blot analysis (1; data not shown) and immunohistochemistry (Figure 2) were used to confirm array data. Western blotting
results were consistent with array results for 7 of the proteins
identified, but not for 2 of them. One of the 2 proteins (Hsp90)
also failed to show overexpression when analyzed by immunohistochemistry (Figure 2, Panel A).
MCL 1
MCL 2
Rb2
KU80
Ab Microarray 500 Analysis
of Mantle Cell Lymphoma
The Ab Microarray 500 was used in the proteomic analysis of
mantle cell lymphoma (MCL; 1). Seven samples were analyzed
and compared to a control sample. Six of the samples showed
13 proteins that were overexpressed when compared to the control,
and one sample displayed an inverted expression profile (Figure 1).
The latter sample was reanalyzed and the same results were observed.
Tonsil
Paxillin
Bcl-x
Figure 2. Immunohistochemistry was used to analyze false positive
and false negative results. Paraffin-embedded tissue biopsies available
from the same patients were analyzed using antibodies present on
the array in order to test for false positives (Panel A) and false
negatives (Panel B). The Panel A results are consistent with the
antibody array in detecting increased levels of CRIK and MDM2,
and the Western blot in failing to demonstrate elevated Hsp90
levels. The Panel B results did not reveal any false negatives for
the proteins analyzed.
False Negatives
Four proteins that did not show changes on the array were analyzed by immunohistochemistry (Figure 2, Panel B). None of the
4 proteins revealed any changes when analyzed by this method,
proving that the array does not yield false negatives.
Conclusion
Comparing relative expression levels with the Ab Microarray 500
revealed the existence of 3 novel proteins associated with mantle
cell lymphoma. The results demonstrate the capacity of the Ab
Microarray 500 for identifying novel proteins as well as the
importance of confirming antibody array data using an independent method.
References
1. Ghobrial, I. M. et al. (2005) Blood 105(9):3722–3730.
2. Andersson, O. et al. (2005) J. Proteome Res. 4(3):758–767.
Ordering Information
Product
Size
Cat. No.
Ab Microarray 500 Slides
2 arrays
631790
Ab Microarray Express Buffer Kit
each
631795
Clontech Laboratories, Inc. • www.clontech.com
Clontechniques April 2009 1 5