February 25th, 2014
The former “Spectroscopy Resource Center” has merged with the High Throughput Screening Resource Center. To reflect this shift in emphasis, we have a new name, “The High Throughput and Spectroscopy Resource Center”, and will retain the acronym, “HTSRC”. The main differences most users will see is that the 2 NMR’s housed on campus will be now booked via our PPMS online booking system and in the coming years the fee structures will be harmonized. Most importantly, we will strive to provide a higher level of service on the NMR instruments, providing for more readily available consulting and advice, making this technology more accessible to the non-expert. Also, many of the technologies offered at HTSRC can provide complementary answers to address the same scientific questions. Thus the HTSRC users will have access to advice and consulting on a broad array of technologies suited for the measurement of biomolecular interactions, small molecule discovery and analysis.
A new “Lenti/ORFeome” gene library is now available to our users/collaborators. Features are highlighted as follows
- Lentiviral expression vector - allows for transduction of almost any mammalian cell
- CMV promoter drives strong expression of the ORF construct
- Blasticidin resistance - allows for multiplex experiments in cells already expressing Puromycin or Neomycin resistance
- V5 tag - facilitates pull-downs and labeling of expressed ORFs via immunofluorescence
- it is arrayed, so one could cherry pick ORFs of interest to study one by one or to make mini pools of interest.
- It is supplied as glycerol stocks
- Gateway-compatible system, the ORF can be moved around to a different vector if necessary.
screening in small or large pools
- screening in arrayed format (using transfection or lentiviruses)
The TECAN EVO150 is the latest liquid handling instrument installed at the HTS Resource Center. This multi-functional liquid handler is equipped with an MCA384 pipetting head to perform microtiter plate-to-plate transfers in both 96- and 384-well plate formats from as low as 1uL to 200uL. The EVO150 is also built with an 8-channel liquid handling arm called the LiHa. This liquid handling arm offers parallel pipetting from 1uL up to 1000uL. The instrument has high-throughput plate stackers that can handle up to 100 plates, a barcode reader, and a disposable tip washer. With this setup, one can quickly replicate plates, cherry pick and re-array samples, perform automated serial dilutions, and with the center’s assistance, build custom applications. The TECAN EVO150 has replaced the Janus Mini.
As with any of our services, please use the online suggestion box to let us know how we can improve.
October 19th, 2012
We are happy to announce several improvements to the capabilities of the High-throughput Screening Resource Center, which are available to you, and present you with a summary below. Please feel free to contact our staff if you have any specific questions.
Improvements in capability for the analysis of biomolecular interactions
We have installed a Proteon XPR surface plasmon instrument. This instrument is more sensitive (mw> 100 versus 300) than our current Biacore 3000 and has higher throughput, allowing for 36 interactions with controls, simultaneously, versus 4 in the biacore. We are happy to train anyone interested and help in the design of experiments.
A Nanotemper microscale thermophoresis instrument has been installed. This instrument can measure biomolecular interactions (equilibrium binding) in a very small volume (8 ul), provided one of the interacting molecules is fluorescently labeled. As contrasted with fluorescence polarization or Surface Plasmon Resonance, there are no specific limits of molecular weight; however, the binding event must effect the interactions of the biomolecule with the solvent system, through changing mass, charge and/or solvation shell, such that its migration through a temperature gradient is affected. We are happy to train anyone interested in this simple technique and help in the design of experiments.
Improvement in Cellular Assays and Western Blots
A LICOR Odyssey SA is an infrared laser scanning instrument that allows for a quantitative determination of antibody binding to western blots or to fixed cells in a microplate. The instrument is capable of reading 50, 384 well microplates in 90 minutes and can scan two colors simultaneously, thus allowing the introduction of normalization controls in every sample. The system has a much larger linear range than chemiluminescent systems typically used in such applications. This is a very useful instrument for measuring antigen expression in both cells and in purified samples.
Improvement of support capabilities for Chemical Selection and Optimization
Cellular profiling assays: For any compound, we now offer two high-throughput cytotoxicity/apoptosis readouts in three standard cell lines, including alamar blue reduction and ATP production. We also have developed a novel high content cellular growth and survival imaging assay can that can be used to screen, classify and/or, prioritize drugs or compounds. It may also help to accelerate mechanism of action studies.
Structure Activity Relationships by Inventory: For any validated hit compound we can now routinely search a database of 4 million commercially available compounds in order to find analogs for structure activity studies. We can also pick the most likely to be soluble, and cell permeable based on Accelrys software algorithms. We can also perform reasonable predictions of metabolism as a starting point for selection of analogs.
Improvements and analysis of the HTSRC compound library
In order to gauge the diversity of our library, we have used Pipeline Pilot chemical analysis software to perform a principal component analysis using molecular fingerprint descriptors. We have compared our library to a comprehensive eMolecules database of 5.6 million commercially available screening compounds. Over 79% of the variance of these structures can be described with the “diversity space” plotted in the below figure, and the scatter in the plot suggests that the Rockefeller University library (170,000 compounds, black spots) is reasonably diverse as compared to the large comprehensive catalogue of commercially available screening compounds (5.6 million compounds, beige spots).
Along with all of the Rockefeller Resource Centers, we have implemented a “suggestion box” on our website that will allow you to place suggestions. If you have any for us, we encourage you to use it!
August 17, 2011
We would like to inform you of some of the latest developments and improvements in the screening center. Most notably, we have dramatically improved our capabilities in the field of “high-content screening.” This type of screen involves collecting large numbers of images of cells and uses complex image recognition algorithms to quantify the effects of compounds on the cells. As you may know, we recently moved to a new and improved space in the ground floor of Bronk building (room 208-216) and now support the Circular dichroism and Surface Plasmon Resonance Instruments (Biacore 3000) formerly housed in the Spectroscopy Resource Center. With the new capabilities, many of the projects we are now supporting are not necessarily compound screening projects, but also include various assay development and biomolecular interactions projects. We really appreciate your feedback as to any improvements we can make. A summary follows.
Biotek synergy H4 with Biostack Plate Feeder
A multi-purpose high-speed microplate reader with dual monochromators which can allow the user to dial in the particular wavelengths of interest. Can read alphaLisa, TR-FRET, assays. Equipped with in-line injection port which allows for rapid kinetic analyses. Capable of reading a 384-well plate in 2 minutes.
Molecular Devices Image Express Velo Laser Scanning Cytometer
This two color (488nm and 534nm) laser-based scanning imager can image 384 wells in 5-10 minutes. The system is equipped with a Twister II robot arm such that 40 plates can be processed unattended. The software can score fluorescent intensity per cell , number of cells, surface area of cells, ellipticity of cells in up two three individual emission bands.
Molecular Devices ImageXpress Micro Fluorescence Microscopic Imaging
384 wells in 15-40 minutes fed with a Thermo CRS robot arm driven by POLARA scheduling software. MetaXpress software can analyze and score imaging for morphological and subcellular events, such as translocation, spot formation and process outgrowth. The detection system is based on a fluorescence microscope with automatic laser focusing
The compound library now contains 171,000 small organic compounds and is expanding. 3700 Newly purchased compounds from LifeChemicals in Ukraine are being delivered. The compounds are chosen to increase the structural diversity of the library, while maintaining a good quality with respect to drug-likeness and lack of potential for unspecific binding interactions. The complete library has now been re-formatted into 384-well plates and a “diversity” library has been created. Thus, users interested in screening less compounds to begin with can “sample” the library based on diversity algorithms.
Using vendor compound data from over 1 million compound database, we have now begun to “annotate” the library with structural themes and warning features. All screening data is entered into the database such that “frequent hitter” classes can be identified.
The following instruments and associated techniques are now available and training is supported: a Fourier transform infrared spectrometer (FTIR), a circular dichroism spectrometer (CD), a surface plasmon resonance (Biacore 3000) detector (SPR), a scanning spectrofluirometer and an isothermal calorimeter (GE AutoITC 200).