Services

Details for our services are divided into two areas. For more information about Molecular Screening and Protein Expression services click one of the following links:

Molecular Screening Services
Protein Expression Services

Molecular Screening Services

The facility provides a range of specialized services critical to high-throughput screening biology. A critical aspect of the Facility’s services is an intensive consultation between prospective users and the facility’s managing and scientific directors. These interactions occur throughout the duration of a project, from target justification to selection and optimization of the most appropriate assay technologies, to data analysis and interpretation of high-throughput screens, and finally, post-screening advancement.

For details about specialized services click one of the following links:

Library Management
Assay Development
High-throughput Screening
Post-Screening Studies
Data Handling and Analysis

Specialized Services

Library management

Laboratory staff is responsible for the collection, handling, and formatting of libraries for screening applications.  In addition, the laboratory staff handles all library transfer procedures, including pinning compounds to assay plates and “hit” picking.

Assay development

Guidelines to developing a miniaturized bioassay for High-throughput screening
Plate type
Assay volume
Optimization of assay variables
Quantitative assay evaluation

The overarching goal for Assay Development focuses on the timely delivery of robust and well validated biological assays ready for implementation in screening experiments. Users are encouraged to contact laboratory staff as early as possible and remain actively engaged throughout the entire process.  The laboratory staff provide:

     - Expertise in cell biology, molecular biology, enzymology, biochemistry, pharmacology, and microbiology.

     - Facilities for propagation of bacteria, yeast, insect cells, mammalian cells, and viruses (i.e. baculovirus, retrovirus).

     - Expertise in expression and purification of recombinant proteins.

     - Equipment and expertise for maintenance, quality control and molecular characterization of normal and engineered cells for cell-based assays under standard biosafety containment up to and including BSL-2.

     - Experience with a variety of biochemical, cellular, immunological, and image based assays using an array of equipment compatible with bench scale or HTS scale experiments.

Guidelines to developing a miniaturized bioassay for High-throughput screening

The process of assay development includes the identification of assay types for screening and determination of structure-activity-relationships, development of assay reagents, optimization of assay parameters for signal intensity, signal window, and precision, adaptation to automation, scalability, and quantitative assessment of the assay's fitness for screening. Assay parameters to optimize assay's include sensitivity to enable identification of compounds with low-potentcy, precision of biological response between wells and plates, accuracy of positive and negative control compounds with known pharmacology towards the intended target, and economic feasibility.

Plate type

Depending on the method of detection used by an assay, different types of plates are recommended. For a list of plates and suppliers, please consult with the screening laboratory staff. 

Assay volume

For high-throughput screening, a biochemical or cell-based assay must be adapted to a microtiter plate format.  Assays are most commonly performed in 96-, 384-, or 1536-well assay plates.  This laboratory recommends that assays be miniaturized to a 384-well plate.  Assay volumes in 384-well plates range from 5 µl (in low-volume plates) to 100 µl (in standard plates). Due to inaccuracies in small-volume pipetting and the potential risk of spillage and/or cross-contamination at the high end, it is recommended that investigators use an assay volume of 30-50 µl in standard plates.  

Optimization of assay variables

Reagent stability:  It is important to test the stability of assay reagents for standard storage and handling conditions in order to define procedures that minimize the loss of activity.  In addition, it is essential to determine the effects of modifications in preparation procedures and lot variation on assay signal and variation.  These data are usually determined by subjecting the handling of one assay reagent to different conditions for various times prior to addition to the assay.

Reaction stability:  It will be essential to determine the stability of the assay’s signal window and precision with respect to incubation time of various steps in the assay protocol.  These data are expected to provide information that greatly aid in defining logistical questions related to screening and tolerance of the assay to potential delays encountered during screening experiments. 

DMSO sensitivity:  Small molecule libraries are commonly formatted at fixed concentrations in 100% DMSO.  Therefore, it is important to determine the compatibility of the assay’s response to a range of DMSO concentrations.  It is recommended that these studies are done early in the development process, as replicate plate experiments to assess an assay’s fitness for screening should be done in the presence of the anticipated final DMSO concentration.

Quantitative assay evaluation

The Z´-factor calculation provides a widely accepted method to quantitatively assess the fitness of specific assay conditions (Zhang et. al. 1999).  The Z’-factor is a unit-less numeric value that takes into account both the signal window and precision of measuring the maximum and minimum control signals in replicate wells.  Newly developed assay conditions are considered validated for high-throughput screening after a replicate plate experiment has demonstrated the assay conditions meet minimum criteria for acceptance. Each experiment should be performed on at least 2 full 384 well plates where 1⁄2 of the wells contain minimum signal controls and 1⁄2 of the wells contain maximum control signals, set up on 3 independent days starting from scratch. For previously established/tested assays, a single day replicate plate experiment will be sufficient. This assessment will produce a statistically significant data set for evaluation. 

To quantitatively rank assay conditions, calculate Z´ from the data collected, using:

SD + = positive control standard deviation

SD - = negative control standard deviation

Ave + = positive control average

Ave - = negative control average

Self Calculating Z-factor MS Excel workbook.

The following table is useful for evaluating the potential performance you might expect from these defined assay conditions during screening experiments.

1 > Z´ > 0.9 An excellent assay

0.9 > Z´ > 0.7 A good assay

0.7 > Z´ > 0.5 Hit selection will benefit significantly from any improvement

0.5 = Z´ The absolute minimum recommend for high throughput screening

This table represents general guidelines that are widely accepted in the academic screening community. If optimization is needed, different assay conditions should be compared and ranked by their Z´-factor values until suitable conditions are found.  Please note that screening results rarely achieve the high quality levels seen during the pilot phase using defined controls.

Ji-Hu Zhang, Thomas D. Y. Chung and Kevin R. Oldenburg (1999). A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen 4:67-73.

For a very thorough discussion of guidelines to developing specific assay types for HTS please see http://www.ncbi.nlm.nih.gov/books/NBK53196/.

 

High-throughput-screening

Pilot screening 

After establishing an optimized assay protocol that meets acceptable criteria in a replicate plate test, a pilot screen of 2,000 to 3,000 compounds from a reference library with a high-degree of pharmacophore content is recommended.  The purpose of the pilot screen is to assess the behavior of the assay conditions and automation procedures you have optimized during assay development under actual screening conditions.  In addition, this pilot screen will be used to establish criteria for ‘hit’ conditions and selection.

Production screens 

The facility has the capability to process upto ~100 microtiter plates per day depending on the assay type and number of steps. Staff will work with investigators/users to determine screen logistics (time considerations, libraries, amounts/volumes of reagents, etc). For newly developed assays, it is recommended that the assay conditions be tested in a pilot-scale screen (see above).  For previously developed assays, demonstration of acceptable criteria in a single day replicate plate procedure will be sufficient to proceed with a production screen.  When performing a production screen, it is preferable to process as many plates as possible during usage of the equipment and laboratory staff time.

To schedule any screen, contact Joseph Salvino.  Do not make arrangements with staff of the facility.  Please plan to schedule your screen well in advance of the date you would like to start your experiment to eliminate scheduling conflicts and to ensure that the libraries are properly formatted.

Only screening facility personnel are permitted to handle library stock plates, and thus they perform all compound transfers from library plates into assay plates. Screening facility personnel will not be responsible for conducting any other parts of a screen unless agreed upon prior to the initiation of the screen, and will be charged as assisted use.

The laboratory conducting the screen is responsible for purchasing all plasticware to be used for the screen, this includes pipette tips, plates, and any other materials specific to your screen (reagent troughs, plate seals, tissue culture reagents, assay reagents, labels for barcoding etc.). The screening laboratory has also negotiated some bulk discounting on plates and pipette tips for the liquid handling unit, and therefore, investigators can purchase these items from the screening facility at cost.

Post-screening studies

The laboratory staff will assist users with cherry-picking requests of candidate ‘hits’ from primary screens, preparation of plates with serial dilutions for IC50/Ki measurements, and development of orthogonal assays and counter-screens to confirm the accuracy and rank order potency of identified compounds. 

Data handling and analysis

In conjunction with the Wistar Cancer Center Bioinformatics core, infrastructure is being established with a broad range of capabilities for data handling and storage. The goal is to provide the ability to analyze biological data sets from screening experiments and extract structure-activity relationship (SAR) information to assist prioritization of hit follow-up. We anticipate these goals being achieved by implementing commercially available software applications and custom designed applications developed by staff in the Bioinformatics shared facility.

Planned capabilities:

 - Compound registration

 - Biological assay registration

 - Statistical analysis of biological assay data

 - Heterogeneous database querying and reporting

 - Clustering

 - Similarity-Diversity analyses

 - In Silico profiling

 - Structure-based docking and scoring

 - Designing and enumerating virtual libraries

 

Protein Expression Services

The Protein Expression Facility operates as a fee-for-service laboratory committed to providing excellent technical assistance in the following areas:

Recombinant Plasmid DNA Engineering
Recombinant Protein Production via Baculovirus Expression Systems (BVES)
Recombinant Protein Production in Prokaryotic Systems
Recombinant Protein Purification
Retrovirus Vector Production

Recombinant Plasmid DNA Engineering

Timeline:

~2-3 weeks

Required Material*:

5 µg of plasmid containing gene of interest 
Genbank accession number
Complete vector sequence or map

Deliverables:

At least 100 µg of ready-to-use recombinant plasmid DNA containing the gene of interest

The facility staff will assist users in designing a cloning strategy to engineer an appropriate recombinant plasmid DNA that meets the specific goals of the project.  The facility supports conventional, ligation independent, Gateway, and site-directed mutagenesis cloning technologies.  The nucleic acids (e.g., cDNA, promoter, shRNA, etc.) are isolated from a customer-supplied plasmid DNA(s) and subcloned into a new vector of choice.   Newly derived plasmid DNAs are authenticated by restriction endonuclease digestion, DNA sequencing, and prepared for subsequent transfection based applications. View a list of available expression vectors.

*Note: Customers are responsible for providing the correct information regarding nucleic acid sequence and plasmid DNAs for subcloning projects. The user will be responsible for all costs incurred during cloning projects where the sequence of the supplied plasmid does not correspond with the sequence provided.

Recombinant Protein Production via Baculovirus Expression Systems (BVES)

Baculovirus expression systems (BVES) represent an alternative approach to produce large amounts of properly folded and functional recombinant proteins. The benefits of protein expression with baculovirus include the virus being able to accommodate large inserts, eukaryotic post-translational modification, enhanced protein folding and function, high expression levels, easy scale up with high-density suspension culture, and safety. The Facility has >20 years of experience in preparing baculovirus vectors from nearly all commercially available BVES. View a list of available vectors.

Investigators have the option to provide the facility with previously constructed baculovirus transfer vectors, have facility staff engineer the plasmid DNA construct, or to provide the facility with small volumes of previously prepared virus stock.

Available services

Preparation of high-titer baculovirus stock

The generation of recombinant baculovirus is achieved via one of two approaches. Spodoptera frugiperda (Sf9) insect cells are cotransfected with the transfer vector plasmid DNA containing the foreign gene to be expressed and baculovirus DNA from Autographica californica nuclear polyhedrosis virus (AcNPV)-Baculogold method. Alternatively, insect cells are transfected with a recombinant bacmid DNA recovered by transposition of the transfer vector DNA in E. coli cells (DH10Bac), the so-called Bac-to-Bac™ (Invitrogen-Gibco/Life Technologies) method. Investigators have the option to provide the facility with previously constructed baculovirus transfer vectors or have facility staff engineer the plasmid DNA construct. View more information about baculovirus expression systems.

Baculogold (Pharmingen, Sigma)-3 weeks

  • Transfection of insect cells (Sf9) with the recombinant transfer vector (containing gene of interest to be expressed) and baculoviral DNA.
  • Amplification of high-titer recombinant baculovirus stock (250 ml-P2).
  • (Optional) virus titering.

Bac-to-Bac (Invitrogen)-3 weeks

  • Transposition of pFastBac transfer vector in DH10Bac, Bacmid preparation, PCR comfirmation of bacmid transposition.
  • Transfection of insect cells (Sf9) with Bacmid DNA.
  • Amplification of high-titer recombinant baculovirus stock (250 ml-P2).
  • (Optional) virus titering.

The BaculoGold™ and Bac-to-Bac™ methods are designed to achieve virtually 100% recombination efficiencies and recombinant protein expression is subsequently evaluated using recombinant virus amplified (without plaque purification) in P2 in insect cells. A single round of plaque purification of recombinant virus from the initial virus production (P1 virus) is optional before virus is amplified in a second passage (P2) for these methods. Recombinant baculovirus derived from all other commercially available baculovirus DNA preparations is produced with 80-90% efficiency and requires plaque purification to remove parental virus.

Timeline:

~3 weeks

Required Materials:

5 µg of recombinant baculovirus transfer plasmid containing gene of interest.

Deliverables:

250 ml of high-titer (≥1 x 10pfu/ml) recombinant baculovirus stock solution in serum free medium ready to express the protein of interest

Amplification of high-titer baculovirus stocks

 The purpose of this service is to prepare high-titer (>108 pfu/ml) baculovirus stocks in serum free medium for preparative scale productions of recombinant protein in insect cells. Titration of the final high-titer stock is an optional service.

Timeline:

2~3 weeks

Required Materials:

At least 5 ml of recombinant baculovirus stock.

Deliverables:

Specified quantity (in ml) of high-titer recombinant baculovirus stock solution ready to express the protein of interest.

Analytical scale productions for optimization of protein expression

The purpose of this service is to aid investigators in assessing the optimal conditions for protein production. The results can be used to determine optimal conditions for protein production. There are numerous examples where the integrity, stability and biological activity of recombinant proteins vary with time after infection. Therefore the recommendation of the facility is that each new baculovirus for a protein be extensively characterized in analytical scale productions before proceeding to large-scale productions.

This service entails the infection of a 100 ml suspension culture of upto three insect cell lines [Sf21, Sf9, and High Five (T.ni)] using a high-titer baculovirus stock. Cells or conditioned media (for secreted proteins) are harvested at 24, 48, and 72 hours post infection. Harvested samples are analyzed for expression of the recombinant protein by western blot. Investigators can complete the Western blot analysis or have the facility staff complete the analysis for an additional fee (customer must provide appropriate gene specific antibodies, if desired).

Timeline:

1 week

Required Materials:

At least 5-10 ml of high-titer recombinant baculovirus stock.

Deliverables:

Cell pellets (or conditioned medium for secreted proteins) from 10-20 ml of infected insect cells ready for expression/solubility analysis by western blotting or small scale purification. A sample of uninfected insect cells is also provided as a negative control.

Preparative scale productions

Expression of recombinant protein under optimized conditions in high-density (1-2 x10cells/ml) suspension cultures (50 ml-1L) of Sf9, Sf21, or Trichoplusia ni (commonly referred to as High Five) cells at a multiplicity of infection (MOI) equal to 1-2. We recommend use of High Five cells for production of secreted proteins.

Timeline:

1-2 weeks

Required Materials:

Predetermined, optimized protein expression conditions (cell line, MOI, infection time).

 

1 liter: at least 25 ml of high-titer (>10pfu/ml) recombinant baculovirus stock.

2 liter: at least 50 ml of high-titer (>10pfu/ml) recombinant baculovirus stock.

4 liter: at least 100 ml of high-titer (>10pfu/ml) recombinant baculovirus stock.

Deliverables*:

Cell pellets ready for protein purification or in the case of a secreted recombinant protein, the customer will receive the supernatant

*We do not guarantee the yield of purified protein/volume of culture since it changes from protein to protein. To achieve a certain yield, additional culture volumes may be required.

**We offer discounts for multi-liter productions done simultaneously.

Recombinant Protein Production in Prokaryotic Systems

Use of prokaryotic expression systems offers an economical method to achieve production of large amounts of recombinant protein. The Facility provides technical support for production of recombinant protein in bacteria.

The core maintains a repository of inducible bacterial expression vector technologies. (View a list of available expression vectors.) This collection of expression vectors allows for fusion of epitope tags (alone or in combination) to the protein of interest (GOI), including GST (Glutathione-S-transferase), 6 histidines (6His), SUMO, FLAG, or maltose binding protein (MBP) at the NH3- or COOH-terminus of a protein. High level protein expression (analytical or preparative scale) is achieved by IPTG induction of T7/T5 RNA polymerases in high-performance Epicuran Coli (e.g. Rosetta (DE3)).

Available services

Optimization of soluble protein expression

The purpose of this service is to optimize the expression of soluble protein. Because some proteins fold poorly or incompletely when over-expressed in E. coli, the Facility uses several strategies to adjust variables that account for inclusion body formation such as N-terminal protein fusion to soluble tags (e.g. GST, SUMO, MBP), culture medium and aeration, growth temperature (18°C-37°C), expression vector characteristics including promoter activity, and use of bacterial strains engineered to express underrepresented tRNA species during production.

Bacterial expression vectors containing a gene of interest are transformed into a strain of bacteria (e.g. BL21) optimized for recombinant protein expression. An analytical scale (100 ml) induction of protein expression is used to determine the extent of the recombinant protein’s solubility under the defined experimental conditions by SDS-PAGE and Coomassie stain.
Investigators have the option to provide the facility with previously constructed baculovirus transfer vectors, have facility staff engineer the plasmid DNA construct, or to provide the facility with small volumes of previously prepared virus stock.

Timeline:

<1 week

Required Materials:

1-2 µg of bacterial expression plasmid containing gene of interest.

Deliverables:

  • Cryopreserved glycerol stocks of transformed bacterial strain(s)
  • Experimental reports and QC data

 

Preparative production

The laboratory has the capacity to express recombinant proteins in bacteria upto 12 L per a day.

Timeline:

<1 week

Required Materials:

1-2 µg of bacterial expression plasmid containing gene of interest or, cryopreserved glycerol stock.
Induction instructions (e.g. temp., time, IPTG conc.)

Deliverables:

  • Induced cell pellets
  • Experimental reports and QC data

 

Recombinant Protein Purification

The Facility provides analytical and preparative scale, one- or two-step purification of recombinant proteins expressed in bacteria and baculovirus infected insect cells. The expectation is to provide highly purified recombinant proteins for use in structure-function studies, crystallographic efforts, assay development for high-throughput small molecule screening, custom antibody productions, and peptide identification of macromolecular protein complexes. Since most recombinant protein expression in bacterial and baculovirus systems takes advantage of epitope tags for monitoring expression, proteins are purified from soluble extracts using appropriate affinity matrices (e.g., Ni2+-/Co2+-agarose, GSH-sepharose, amylose-agarose, anti-FLAG/HA agarose) that allow for selection of specific tags (e.g. 6His, GST).

When necessary, a second purification step (e.g., affinity, size exclusion chromatography, etc) is included to ensure maximum purity of the recombinant protein. The core cleaves epitope tags, when requested, and the recombinant protein will be further purified from tags and proteases. Purified proteins are dialyzed into appropriate buffers and concentrated to desired concentrations.

Timeline:

~1-2 weeks

Required Materials:

Cellular source of recombinant protein expression

Deliverables:

  • Purified protein dialyzed into desired storage buffer
  • Experimental reports and QC data

Retrovirus Production

The development of retroviral delivery systems for mammalian cells has greatly enhanced the ability of scientists to deliver genetic material to cells of diverse origin in order to study the role of a particular protein or network of proteins in a specific cell type or biological process. The goal of the retrovirus production unit is to assist Cancer Center members with the preparation of high-titer retrovirus (e.g. lentivirus). The centralization of such services provides economy of scale, high-throughput production, increased biosafety, and quality control in the virus preparation process. The Facility provides a variety of services including prepackaged vector preparations, custom vector packaging, and vector titrations.

The Facility’s work with recombinant retrovirus DNA is registered with the Wistar Institute IBC.

Available services

Distribution of prepackaged virus
Custom lentivirus preparation
Vector concentration by ultracentrifugation
Vector titration

Distribution of prepackaged virus

1.  Fluorescent protein tracer: These vectors express fluorescent proteins (e.g. GFP, DsRed2, mCherry) from a strong constitutive promoter (e.g. CMV, EF1a, PGK) and are useful for marking cell populations and to experimentally determine the transduction efficiency of a cell line.

 2. Luciferase vector: These vectors express firefly luciferase from a strong constitutive promoter (e.g. EF1) and are useful for monitoring cell populations in vitro and in vivo.

 3. pLKO shRNA controls:
This vectors serve as controls (e.g., empty, non-targeting, siGFP, siLuc, etc) for experiments using TRC (pLKO.1) shRNA plasmids. 

* All prepackaged vector preparations are VSVg pseudotyped, non-replicating lentivirus and come as filtered, concentrated vector in PBS. Concentration is performed by ultracentrifugation. All vectors, except pLKO-based, are quantitatively titrated using HEK293T cells and percentage of transduced cells determined by flow cytometry. Note: pLKO.1 vectors do not have a fluorescent marker. Therefore, the titer of these vector stocks has been determined by a p24 assay. 

** The Wistar Institute Vector production unit has purchased the above plasmids from Sigma-Aldrich and Open Biosystems and does not profit from the sale of lentivirus in any way. All lentiviral vectors produced by the Wistar Vector Production Unit and distributed to Cancer Center laboratories are for use in research only.

View a list of available vectors.

Custom packaging of vectors

Time:

1-2 weeks

Required materials:

Billing information
Completed submission form
Required amount of plasmid DNA:
15 ml conditioned cell supernatant: 10 ug

30 ml conditioned cell supernatant: 20ug

60 ml conditioned cell supernatant: 40 ug

120 ml conditioned cell supernatant: 80 ug

Deliverables:

Options:
A. 15ml conditioned cell supernatant

B. 30ml conditioned cell supernatant 

C. 60ml conditioned cell supernatant

D. 120ml conditioned cell supernatant 

E. 30ml concentrated supernatant resuspended in PBS

F. 60ml concentrated supernatant resuspended in PBS

G. 120ml concentrated supernatant resuspended in PBS

The facility will prepare non-replicating, VSVg pseudotyped vectors by:

 • Transfecting retrovirus vector, 2nd generation packaging plasmid, and VSVg plasmid into HEK293T cells

 • Collect viral supernatants at 12-24 hr intervals

Retrovirus production services are ordered based on volume of conditioned cell culture supernatant.

Concentration of vector by ultracentrifugation

The Retrovirus Production unit will concentrate (~1000X) vector preparations by ultracentrifugation. All concentrated vectors are provided in PBS as 5-10 ul aliquots.

Vector titration

Vector production unit determines titers of vectors using a quantitative titration protocol on HEK293T cells, if the vector contains a fluorescent marker. Please do NOT request titrations on other cell lines or using alternative methods.

Guidelines for custom lentivirus preparation:

1. To schedule custom preparation of lentivirus, investigators must submit a completed plasmid submission form.

2. Customer billing information and a valid grant number (Wistar investigators only) or PO# (all external users) must be provided prior to the initiation of any work.


 3. To address safety concerns, please provide information on the nature of your vector (i.e. promoter, markers, non-replicating) and insert (i.e. select agent, oncogene, immunosuppressant, toxin, etc.). 


4. Please provide the appropriate amount of transfection quality (i.e. Qiagen, Wizard prep, etc.) lentiviral vector DNA, containing your gene of interest, which you want to have packaged into the virus, with supporting submission form. Note: We do not accept DNA minipreps.

 5. Please indicate the concentration of your transfer vector (we prefer a working concentration of 0.5-1μg/μL).


 6. Please indicate the services (volume of cell supernatant, concentration, titration) you are requesting.


 7. Due to variability in packaging of different retroviral vectors, production of a minimum titer cannot be guaranteed. Whenever possible, newly produced vectors should be titrated.


 8. It is the responsibility of the investigator to register the intended specific use of recombinant DAN technology (i.e. retrovirus use) with Wistar’s Institutional Biosafety Committee (IBC) as required by the most current NIH Guidelines for Research Involving Recombinant DNA Molecules.

9. Please acknowledge the Protein Expression Facility if viruses are used in the generation of publication data.


Additional Lentivirus Information