High-Throughput Micro-Western Array Service
 
  
  • Introduction
High-throughput research is currently the most efficient research method in the field of systems biology. This technique will allow scientists to observe large numbers of samples and large-scale systematic studies and will provide quantitative data for analyzing. Its use in researches, such as in various cellular signaling network studies, changes of disease pathology, responses with drugs, development, differentiation, etc., becomes very important.
 
Expression and condition (such as whether or not phosphorylation activated) of proteins are the main factors that truly affect the majority cellular physiology, conditions, responses, and behaviors; therefore, conducting high-throughput analytical method will advance scientists’ understanding of signaling transduction of various cellular networks. Laboratories have used Western blots for measuring protein expressions in their interested network studies, through running gel electrophoresis and soaking in specific antibodies, to quantitatively analyze protein changes in the cells. But this method is limited by a need for large amounts of sample and expensive antibodies, and the inability to measure amount of proteins at a time. With hundreds or even thousands of proteins involved in cellular networks, scientists were restricted to observing only a small fraction of protein activity with each experiment.

 
 
       
Dr. Chih-Pin Chuu, Assistant Investigator, Institute of Cellular and System Medicine at NHRI, had participated in the research and development of new high-throughput western blotting technique when he was a postdoctoral fellow in Dr. Richard B. Jones’s lab at the University of Chicago. This technique, described as the micro-western array, combines the specificity of the popular “Western blot” protein array with the large scale of DNA microarrays. A pre-printed micro-western array 96-well gel essentially comprises 96 miniature Western blots, and each miniature Western blot contains 1 protein marker and 6 samples. In contrast with dot blot or antibody array, this micro-western array technique can separate proteins with different molecular weight to avoid cross-reaction, and the results will be similar as using the traditional western blot; and only 0.2 μl of antibody and 50 nl of sample are needed for each micro-western blotting.
 
The arraying runs for 4 hours each time and can print a maximum of 2 gels, which contain 192 individual Western blots and a total of 1152 sample spots. Each micro-western blot can use one kind of 0.2 μl antibody; therefore, a total of 192 sets of antibodies can be used in one experiment. Just similar to traditional western blotting, the whole process of this micro-western array including transfer, blocking, washing, and soaking the 2ndantibody only takes 2 days, and furthermore, only 2-3 days are needed for analyzing data. Therefore, in the result of work load contrast: a person runs experiments using micro-western arrays in one week is about using the traditional Western blots for 24 weeks, which is also about the half-year work load. This new approach allows scientists using a tiny amount of sample and antibody to analyze the changes of a large amount of different proteins and is most suitable for using on the studies of the signaling transduction pathway changes of stem cells, cancer cells, and tissue development.
 
 
        

  Nature methods 7, 148(2010) 

      

 

 

Since almost all laboratories would use Western blots, this new method will assist laboratories in various interested research fields. This system has been successfully set up in the Protein Chemistry Core Laboratory, and the service is run by the Core’s trained operator. The instrument, Arrayer, is accessible by the operator only, and the Core only provides micro-western array service. In addition to the 6-sample mode, the Core has improved the printing patterns and can print 15 samples and screen 48 antibodies at one time, more suitable for using on the clinical samples or drug screening analyses.
 
  • Users 
    Only the NHRI users are eligible for using the service.
    Service costs may be charged to PI’s intramural or NSC project grants/budget account. 
 
  •  Sample Submission
Please contact Ms. Kang-Lin Chu (ext.33702/33712/33713; email: kanglinchu@nhri.org.tw) at the Protein Chemistry Core Laboratory first. Users can choose to submit samples either in cell pellet or in lysate. If users would like to prepare samples in a lysate form, please use the specific lysis buffer provided by the Protein Chemistry Core Laboratory.
 
To request the service, please go to the Protein Chemistry Core Laboratory website, complete the application form and have it authorized, then submit it with the samples together.
 
  • Service Fees
In accordance with the goal of long-term use of the instrument and providing not-for-profit service, all users will be assessed a minimum cost based on the amount of materials and supplies used plus the total costs of the antibodies that users have requested. If user needs to add any the-Core-provided antibody to the samples, it costs NT$100 per antibody. There is no additional charge for adding any self-provided antibody.
 
 
  • Basic Fees
#
Item
No. of Antibody
No. of Sample
Cost (NT$)
Remarks
1
1 gel, 6 samples/ 96 sets, duplicate (48 Ab)
48
6
2500
 
2
1 gel, 6 samples/ 96 sets, singlet (96 Ab)
96
6
3000
 
3
2 gel, 6 samples/ 96 sets, duplicate (96 Ab)
96
6
4200
 
4
2 gel, 6 samples/ 96 sets, singlet (192 Ab)
192
6
4500
 
5
1 gel, 15 samples/ 48 sets, singlet (48 Ab)
48
15
3300
 
 
 
Users can pay NT$1000 per gel to have the Core to quantitate and analyze the produced raw data. Users may also borrow the Licor disc from the Core to install software on their own laboratory computer for self-analyzing.
 
 
  • Dispute Resolving
If any smear is shown on results, the Core will rerun the experiment with no additional charges. If more than half amounts of antibody images do not show, the Core operator will discuss with the user to confirm sample’s specification and condition, then to decide whether or not the Core reruns the experiment one more time for no charge.
 
 
Result image(s) of test run at present:
 
1.        96-well format:
 
 
 

 

 

 

 

 

 

 
2.        48-well format:

 

 

 

 

 

  

 

公告日:2011/07/14





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