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Protein Binding Microarray Service - FAQS |
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GeneralMicroarray Design
Sample PreparationSample Submission
Array ResultsData Analysis
Additional QuestionsAnswers - GeneralWhat are protein binding- peptide microarrays? Protein binding – peptide microarrays are in situ synthesized peptide microarrays that utilize the µParaflo® microfluidics technology for on-chip synthesis of peptides. Each microarray consists of up to thousands of 8mer (or longer) peptides. These microarrays can be used for peptide mapping, drug discovery research and other applications. Microarray DesignWhat is the array platform used by LC Sciences? These are not spotted arrays! Our peptide microarray synthesis is based on a proprietary µParaflo® microfluidic chip technology developed by our partner, Atactic Technologies. This flexible technology enables fast, on chip synthesis of microarrays when ordered. (vs. an off-the-shelf spotted array) Please see our µParaflo® technology bulletin or the following references 1-2 for further information. 1. Pellois, J. P., Zhou, X., Srivannavit, O., Zhou, T., Gulari, E., and Gao, X. (2002) "Individually addressable parallel peptide synthesis on microchips". Nat. Biotechnol. 20, 922-926. 2. Gao, X., Pellois, J. P., Kim, K., Na, Y. , Gulari, E., and Zhou, X. (2004) “High density peptide microarrays. In situ synthesis and applications”. Molecular Diversity. 8, 177-187. These are completely custom microarrays so you decide what goes on the array. We can synthesize up to 3,918 customer defined sequences on a µParaflo® microfluidics peptide chip. length up to 8mer This service gives you total freedom for the choice of peptides, up to thousands of sequences. Please talk to our customer support about your requirements. What is the cost for adding longer peptides? If any of your sequences are longer than 8 residues (it doesn’t matter how many), there will be a $100 charge each for residue #9 and #10, and a $150 charge each for residue #11 and #12 (count from the longest peptide). How many redundancies of each custom sequence do you recommend? We recommend triplicates although this will reduce the total number of sequences you can add to the array. Do I need to perform replicate arrays for more confidence in my results? We synthesize all sequences in a minimum of triplicate on the chip and our intra chip variability is very low. For single species arrays, more replicates can be added upon your request. Further, because our chips are based on the µParaflo® technology, the spot uniformity is excellent both within the chip and from chip to chip. The decision of how many replicate chips to run is up to you. Generally, customers will run a single chip and make an assessment to determine if experimental design is OK or needs to be altered before multiple chips are run to validate results. If you think there might be variability in your sample, you may want to run multiple (3) arrays. Sample PreparationHow much sample do I need to send to LC Sciences? The required protein amount differs and will be determined by protein binding affinity and the objective of the chip assay. Typically, each assay requires about 250 µL of the sample at µM concentration. This is about 0.25 nmol or 25 µg of a protein (Mw 100 kDa). The binding of Kd ~ 1 µM can be detected under the condition. Proteins of higher binding affinities will require less than the amount given and of lower binding affinities will require more than the amount given. Sample SubmissionWhat is required for sample submission? Please complete a sample submission form and email to us prior to sending your sample to us. We cannot begin work on your array without a PO Number or Credit Card Number. See our sample submission page for further information. How do I pack and ship my sample? Please transfer your sample to a 1.5ml microcentrifuge tube for shipment (0.5 ml or smaller PCR tubes can crack when frozen). If you want to seal the tubes or hold them in a rack, please don't use tape (it will crack when frozen), use parafilm. Pack the sample with dry ice in a thermal insulated shipping box. Ship by overnight carrier for delivery the next day. Note: Do not ship samples on Friday as they will sit over the week-end and deteriorate. Wait until the following Monday to ship the package for Tuesday delivery. Mail your package to:
Attn: Protein Binding Microarray Sample Can I send LC Sciences a sample from overseas? Yes, we have no problems with shipments coming from overseas. Array ResultsHow long will it take to get results? We can generally have data back to you about 4 weeks from the date we receive your protein/antibody sample. For each array, you will receive the original and processed microarray scan images, an array layout file, a raw intensity data file in Excel, and a fully processed data file in Excel. Additionally, for each batch of samples, you will receive a Data Summary containing a catalog of data files, images of representative regions of corresponding arrays, and descriptions of specific features of the arrays. The above files will be stored on a CD and will be delivered to you by express mail. Data AnalysisDoes the comprehensive service include data analysis? Yes, full data analysis is included for each chip in the comprehensive service. What is involved in the data analysis and what type of analysis results will I receive? We have in house software for routine array data processing that follows the common practices of DNA array data treatment.3-6. The data are processed in a MS Excel spreadsheet using a program routine that performs raw signal background subtraction using a local regression method (Xiaochuan Zhou, unpublished results; note that the photolithographically fabricated arrays do not have peripheral areas for background values). Data normalization, using a cyclic LOWESS (Locally-weighted Regression) method7 is used to remove system related variations, such as sample amount variations, dye labeling bias, and signal gain differences between scanners, so that biological relevant variations can be faithfully revealed. Detected signals greater than background plus 3 times the standard deviation will be derived; the mean and the co-variance (CV = stdev x100/replicate mean) of each sequence having a detected signal will be calculated. For two color experiments, the ratio (log transformed) of the two sets of detected signals, and p-values of the t-test, will be calculated. Differentially detected signals are generally accepted as true when the ratios of the p value is less than 0.01. References: 3. Ball, C. A.; Sherlock, G.; Parkinson, H.; Rocca-Sera, P.; Brooksbank, C.; Causton, H. C.; Cavalieri, D.; Gasterland, T.; Hingamp, P.; Holstege, F.; Ringwald, M.; Spellman, P.; Stoeckert, C. J., Jr.; Stewart, J. E.; Taylor, R.; Brazma, A.; Quackenbush, J. (2002) Standards for microarray data. Science 298, 539. 4. Quackenbush, J. (2001) Computational analysis of microarray data. Nature Rev. Genet. 2, 418-27. 5. Quackenbush, J. (2002) Microarray data normalization and transformation. Nature Genet. 32 Suppl., 496-501. 6. Sturn, A.; Quackenbush, J.; Trajanoski, Z. (2002) Genesis: cluster analysis of microarray data. Bioinformatics ;18, 207-8. 7. Bolstad, B. M.; Irizarry, R. A.; Astrand, M.; Speed, T. P.(2003) A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19, 185-93 Yes, the data package that we send to you contains sufficient raw data and array layout information for you to carry out you own data analysis. Additional QuestionsCurrently the array chips are not available for separate purchase without the service. Our technology utilizes a microfluidics chip that requires additional liquid handling equipment not available outside our lab. In the future we hope to be providing this equipment so that users may perform their own experiments. We have a very well-trained and knowledgeable staff of scientists here at LC Sciences. If there is some specific experimental work you need done, we would be more than happy to put together custom service package designed specifically for your research needs. For other questions please send us an email at service@lcsciences.com or call us at 1-888-528-8818
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