Frequently Asked Questions


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Targets

What types of proteins are targeted by this project?

The goal is to develop validated capture reagents to human proteins involved in gene regulation, particularly transcription factors and chromatin associated proteins.

Are there capture reagents for all the proteins on this list?

Yes. All proteins visible on this portal have capture reagents, primarily mouse and recombinant antibody-like molecules.

What is a target?

As defined by this program, a target is a protein. A capture reagent "targets" (binds to) a protein.

What is the difference between a protein and antigen?

Proteins we work with usually have multiple domains (antigens) that can be targeted separately. Therefore, there can be more than one antigen for each protein that can have an available capture reagents.

What protein fragment do the reagents recognize?

Protein structural domains are usually selected as the antigens. The sequence of each antigen is available by clicking on an antigen identifier.

Can I find out what proteins are in your pipeline?

We plan to provide this feature in the next release of the portal.

If a protein is in your pipeline, can I sign up for an alert when a reagent is ready?

We plan to provide this feature in the next release of the portal.

Can I nominate a new protein target or increase the priority for a target?

We plan to provide this feature in the next release of the portal.


Reagents

What is a capture reagent?

A capture reagent is considered a unique binder to a specific antigen. It can be an antibody or recombinant antibody-like molecule.

What is a recombinant antibody-like molecule?

Recombinant antibody-like molecule is a human antibody Fab fragment that has been engineered using phage display mutagenesis to bind to the target protein with high affinity and specificity.

What types of reagents are you generating?

We are primarily generating monoclonal antibodies from mouse hybridomas and recombinant antibodies from phage-displayed fragment antigen-binding (Fab) libraries.

How do I order a reagent?

Reagents are being distributed by the DSHB (dshb.biology.uiowa.edu) and the DNASU plasmid repository (dnasu.org) at minimal cost or by CDI (cdi-lab.com). The portal provides links to order from distributors.

How much do reagents cost?

A goal of this program is to distribute all reagents at minimal cost. Each distributor sets its own prices.

Are there any restrictions on the use of these reagents?

Restrictions may vary depending on the distributor. The DSHB restricts the commercialization of purchased reagents or providing to a second party. For DNASU, depositors must sign an agreement, but there is no restriction for recipients. CDI generally has no restrictions for the research market but requires further licenses for commercial purposes including applications as diagnostics or therapeutics.


Search

How do I search for a target?

The easiest way is to use the HGNC gene symbol for your protein. A search panel on the left permits you to search identifiers that include HGNC symbols and aliases, UniProt ACs and IDs, protein names, Entrez Gene IDs, OMIM IDs, and others. The default search is case insenstive for identifiers that begin with the search term; searches may also be for strings contained within an identifier or for exact matches. Searches with multiple space-separated terms return binders for proteins that match at least one term (OR logic).

Other than the HGNC gene symbol, how can I search?

If you don't know the gene symbol, UniProt (uniprot.org) and HGNC (www.genenames.org) provide search and mapping. We already include most popular gene identifiers. For Ensembl, however, only transcript IDs (starting ENST) rather than gene IDs (starting ENSG) are supported.

Can I search by sequence rather than by ID?

In our next release, we plan to provide the ability to search for antigen sequences similar to your protein query. This can be useful if your target is a non-human protein with a human homolog.

Can I search by other criteria?

For disease-related targets, you can search by OMIM ID.


Validations

What validations are performed on reagents?

Types of validation include Western blot (WB), immunoprecipitation (IP), spiked IP, IP followed by mass spec (IP-MS), luciferase immunoprecipitation systems (LIPS), immunofluorescence (IF), competition ELISA, surface plasmon resonance (SPR), ChIP-Seq, and protein microarray.

ChIP

Chromatin immunoprecipitation followed by DNA sequencing (ChIP-Seq, or ChIP) assesses the performance as pulldown reagent for ChIP-Seq using a variety of human cell lines, including cell lines used by the ENCODE consoritum.

Competition ELISA

Competition ELISA is an enzyme-linked immunosorbent assay (ELISA) that uses competitive binding to antigens in solution vs. immobilized to determine the dissociation constant (K_D) for a capture reagent.

Immunofluoresence (IF)

Immunofluorescence (IF) assesses the performance of the binder as an immunofluorescence reagent.

IP

The immunoprecipitation (IP) validation assesses the ability of the reagent to immunoprecipitate targets from cell lysates.

IP-MS

Immunoprecipitation followed by mass spectrometry (IP-MS) identifies the masses of proteins pulled down by the reagent. These masses are then analyzed to assess whether the antibody has sufficient affinity and selectivity to pull down the intended target and potential co-complex members.

What is the protein microarray validation?

The protein microarray uses an array of spotted human proteins to ensure that a reagent is monospecific to a single human target, with negligible off-target activity. It provides quantitative measures of affinity (A-score) and selectivity (S-score).

Spiked IP

Spiked IP generates a titration curve of the capture reagent against the target to provide a dissociation constant (K_D).

SPR

Surface plasmon resonance (SPR) uses electromagnetic waves to measure binding of reagents to targets. It provides quantitative on-rates and off-rates, which can be combined to also calculate the dissociation constant (K_D).

Western Blot (WB)

The western blot (WB) provides a qualitative measure of selectivity by assessing the ability of antibody to generate a band at the expected molecular weight of the target in a complex mixture, generally a cell lysate.

Is each validation performed on each reagent?

No. Validations vary and are specific to production sites. Each reagent, at a minimum, must undergo a primary validation (affinity or specificity) and secondary validation (some form of immunoprecipitation: IP-MS, IB-WB, ChIP-Seq).

How can I see validation results?

Validation results are provided in a data sheet for each reagent. Additional data, including raw data for protein microarray and ChIP-Seq, will be available from the production labs directly.


Credits

How is this project funded?

Protein Capture Reagents is a program funded by the NIH Common Fund.

What groups are part of this effort?

The antigen production center is located at Rutgers, and antibody production centers are at Johns Hopkins University/CDI Labs, and The Recombinant Antibody Network composed of labs from The University of Chicago, University of Toronto, and University of California at San Francisco. See http://commonfund.nih.gov/proteincapture for more information.

How do I acknowledge use of reagents?

Please acknowledge the NIH Common Fund's Protein Capture Reagents Program (http://commonfund.nih.gov/proteincapture) and the distributor.