Finding the Best Capture Reagents

As capture reagents, monoclonal antibodies are the most widely used reagents for specifically detecting and quantifying proteins due to their very high specificity. However, development of monoclonal antibodies is time-consuming and expensive. In addition, many antigens prove to be non-immunogenic or extremely toxic, and therefore cannot be used to generate antibodies in animals. Furthermore, the large size of monoclonal antibodies (150 kDa) may limit their use in cases where more than one binding reagent competes for space to recognize closely juxtaposed epitopes. These limitations could arguably be the biggest hurdles to using monoclonal antibodies as capture reagents for a systematic study of the complete human proteome or for clinical applications of advanced proteomics.

Therefore, alternative capture reagents with high specificity, high affinity, and flexible size and structure that can be easily and cost-effectively produced are urgently needed in order to accelerate proteomic research. Single-chain variable-fragment (scFv) antibodies have been commonly used as alternatives in this regard. scFv is comprised of only the light chain and heavy chain variable regions connected by a peptide linker and with a molecular weight of 27 kDa. Since scFv retains the antigen-binding site of the variable regions, it inherits the specificity of an intact antibody and affinity. In addition, scFv can be easily expressed in yeast or in E. coli with yields in milligrams per liter. scFv can be linked to Fc of desired species specificity and maintain binding properties. If necessary, there is also the option of converting scFv into other antibody formats such as Fab or full IgG by simple cloning steps. The converted antibodies can also be efficiently expressed and purified in yeast or E. coli.

More recently, single domain antibodies that exist in nature were discovered that can be as small as half the size of scFv, and judging from the available data, superior in binding capabilities to scFv or even traditional IgG antibodies. This type of affinity molecules, termed VHH isolated from camelid animals or nurse shark, can be highly expressed in E. coli, linked to a fluorescent protein marker, or chemically conjugated to HRP or other signal generating moieties through a one step reaction.

Surface Bind gDNA for retrieving genomic DNA from Allele-in-One mouse tail lysate.

15% off One step Genotyping (mouse tail lysis and PCR kits), use code 011011MT at shop.allelebiotech.com

Tags: affinity reagents, capture molecules, human proteomics, IgG, nAb: Camelid Antibodies, nAbs, nano antibodies, nanobodies, proteome, scFv, VHH, VHH antibody chromotek

New Frontiers for Research Tool Development in the New Year

Allele Biotech's Green Crystal Ball

Optogenetics
Chosen as the Method of the Year 2010 by Nature Method and mentioned in a number of year-end recaps, this is a technology that allows the use of light to precisely (at least in a temporal sense) control engineered proteins within a targeted cell population. For example, by introducing light-activated channelrhodopsins into neurons, one can use a pulse of light to initiate a movement of ion across the cell membrane. The technology, first reported in 2005 then made headlines as a major impact on neurosciences since 2007, is now being combined with other components in controlling a broader array of biological events, such as DNA binding, enzyme activities, etc. Looking forward, a few areas will be more than likely the frontlines of moving optogenetics into more labs:

Additional combinations: The few known channelrhodopsins and their fast growing variations will be combined with more “effecter” domains to control different events. The challenge will be to find ways to use the structural changes or any responses channelrhodopsins have to stimulating lights in order to trigger a reaction in the associated effecter domain.

Tracking mechanisms: A platter of fluorescent proteins (FPs) will be used as an independent tracking method to follow cells being targeted. FPs that have optical spectra that do not interfere with the optogenetic molecules will be tested and established. In addition, FPs with less toxicity, narrower excitation and emission peaks, and more tolerance to different cellular environment will be preferred and eventually set up as standards.

Delivery tools: To bring the optogenetic reagents into cells like neurons researchers will most likely rely on lentiviral vectors in most cases. Other vehicles such as baculovirus, MMLV-based retrovirus, even herpes virus may find broader applications in this field. Pre-packaged lentiviruses and MMLV-retroviruses already contain optogenetic constructs will become popular products.

VHH Antibodies
The small capture polypeptides based on single-domain Camelid antibodies (nanobodies, nano antbodies or nAbs) and similar VHH domains will become much dramatically more popular this year, judging from the significant increase in demands of the only camelid reagent products, GFP-Trap and RFP-Trap, in 2010. There are a number of NIH initiated programs that aim to find capture reagents that eventually target the complete human proteome. One of the key criteria for the current phase of the relevant NIH Director’s Initiative is ability to co-immunoprecipitate. The Human Proteome Organization (HUPO) recently expressed frustration due to the lack of high quality capture reagents necessary to isolate and identify most proteins. HUPO promotes global research on proteins in order to decode the human proteome. From what we have learned from dozens of publications showing the use of GFP-Trap, VHH molecules pulls down GFP-tagged proteins with unprecedented efficiency and purity. VHH antibodies show strong affinity and specificity, at a level superior or comparable to monoclonal antibodies. In addition, VHH antibodies are increasingly appreciated for their capabilities to recognize concave epitopes by their relatively convex-shaped paratopes. VHH nanobodies are small (~12-15 kD), with a limited number of functionally important disulfide bonds, can be expressed very well in E. coli, and are amazingly stable in extreme denaturing conditions such as heat and acid. They have been shown to be better suited for in vivo and trans-cellular membrane delivery than other antibodies. It should not be surprising that one day in the coming years VHH antibodies will be more dominant than monoclonal antibodies.

Super-Resolution Imaging
One of the goals of developing technologies such as photoactivated localization microscopy (PALM) and related super-resolution imaging (SRI) techniques was to achieve electron microscopy (EM) level resolution without using EM. Now new developments show that maybe combining EM and photoactivable FPs would provide more specific and more detailed morphology. It would be anticipated that more photoconvertible FPs will prove to work well for one type of SRI or another. The event that will bring this technology to nearly every cell biology lab is the improvement and availability of necessary instruments that some companies have already begun to commercialize.

New Product of the Week 010311-010911:

Human let-7b miRNA minigene on lentivirus with RFP reporter, ABP-RP-MILT7BLP

Promotion of the Week 010311-010911:

15% off mWasabi-based organelle markers carried on baculo2mammalian system if order this week (On-Demand products will require about 3-4 weeks for virus packaging after an order is placed). Use code 0103BACFP on fax or email order.

Tags: alpaca antibodies, camelid antibodies, channelrhodopsin, EM, halorhodopsin, optogenetic, PALM, photoactivable FP, photoconvertible FP, STED, STORM, VHH, VHH antibodies

17 Papers Using GFP-Trap, 12 Since 2009

1. MacKay C, Déclais AC, Lundin C et al. (2010). Identification of KIAA1018/FAN1, a DNA repair nuclease recruited to DNA damage by monoubiquitinated FANCD2. Cell 142:65-76.

2. Babiano R, de la Cruz J. (2010). Ribosomal protein L35 is required for 27SB pre-rRNA processing in Saccharomyces cerevisiae. Nucleic Acids Res 2010 Apr 14.

3. Fulcher AJ, Dias MM, Jans DA. (2010). Binding of p110 retinoblastoma protein inhibits nuclear import of simian virus SV40 large tumor antigen. J Biol Chem. 285:17744-53.

4. Taniue K, Nishida A, Hamada F et al. (2010). Sunspot, a link between Wingless signaling and endoreplication in Drosophila. Development. 137:1755-64.

5. Rottach A, Frauer C, Pichler G et al. (2010). The multi-domain protein Np95 connects DNA methylation and histone modification. Nucleic Acids Res. 38:1796-804.

6. Boulon S, Ahmad Y, Trinkle-Mulcahy L et al. (2010). Establishment of a protein frequency library and its application in the reliable identification of specific protein interaction partners. Mol Cell Proteomics. 9:861-79.

7. Schornack S, Fuchs R, Huitema E et al. (2009). Protein mislocalization in plant cells using a GFP-binding chromobody. Plant J. 60:744-54.

8. Fellinger K, Bultmann S, Rothbauer U et al. (2009). Np95 interacts with de novo DNA methyltransferases, Dnmt3a and Dnmt3b, and mediates epigenetic silencing of the viral CMV promoter in embryonic stem cells. EMBO Rep. 10:1259-64.

9. Muñoz IM, Hain K, Déclais AC et al. (2009). Coordination of structure-specific nucleases by human SLX4/BTBD12 is required for DNA repair. Mol Cell. 35:116-27.

10. Webby CJ, Wolf A, et al. (2009). Jmjd6 Catalyses Lysyl-Hydroxylation of U2AF65, a Protein Associated with RNA Splicing. Science. 325:90-93.

11. Rogowski K et al. (2009). Evolutionary divergence of enzymatic mechanisms for posttranslational polyglycylation. Cell. 137: 1076-87.

12. Frauer C, Leonhardt H, (2009) A versatile non-radioactive assay for DNA methyltransferase activity and DNA binding. Nucleic Acid Res. 35: 5402-5409.

13. Trinkle-Mulcahy L et al., (2008) Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. J Cell Biol. 183: s223-39.

14. Rothbauer U, Leonhardt H, (2008) Connecting Biochemistry and Cell Biology with Nanobodies. Zellbiologie aktuell 34: 9-12.

15. Rothbauer U et al., (2008) A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol Cell Proteomics 7: 282-289.

16. Agarwal N et al., (2007) MeCP2 interacts with HP1 and modulates its heterochromatin association during myogenic differentiation. Nucleic Acid Res.35: 5402-5409.

17. Rothbauer U et al., (2006) Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods 3: 887-889.

New Product of the Week 071910-072510: Cre Reporter Cell Line: LoxP-RFP Human Fibroblast, perfect to test our Cre-2A-GFP lentivirus, when cre works, the cell change from red to green.

Promotion of the Week 071910-072510: High Quality dNTP Mix, 10mM, 5 ml, $409 this week $309. Hurry, email to oligo@allelebiotech.com or fax 858-587-6692 by Sunday to save $100 on your lab budget.

Tags: anti-GFP, chromatin immunoprecipitation (ChIP), coIP, GFP, immunoprecipitation, nAb: Camelid Antibodies, Nanobodies, VHH, pull down

BioTechniques Publishes Article on Single Domain Antibodies

Many blogs start by asking “Did you know…” to intrigue you to read along. So here it goes:

Did you know that there are more than 300,000 antibodies that are commercially available? And yes, many antibody companies are still generating more antibodies at ever faster pace and in a more systematic way. There are companies that plan to make peptide or short protein fragments for making antibodies against all human proteins or subproteome, others develop antibodies particularly suitable for demanding assays such as ChIP-CHIP. Government activities such as the National Cancer Institute (NCI)’s Clinical Proteomic Technologies Initiative (CPTI) and the Road Map program under the NIH Director’s Office also set goals of producing comprehensive sets of widely usable, renewable, affinity reagents for clinical cancer samples or the human proteome. Apparently people do not think the 300,000 available antibodies are sufficient for what they do.

Did you know that conventional antibodies commonly used as reagents are ~150kDa in molecular weight and can hardly be used inside live cells? Ulrich Rothbauer, professor in the department of biology at Ludwig Maximilians University, who is working with colleagues to develop tools to study cellular processes in living cells. “These antibodies have to assemble four different chains, two heavy and two light, and they’re assembled by disulfide bonds that cannot be correctly formed in the reducing environment of the cytoplasm. You cannot express such a huge complex molecule in living cells. You can [introduce] them by microinjection, for example, but it’s not applicable for high-throughput cell imaging.” [1] Antibody fragments such as scFv, Fab, and similar derivatives have been developed over the years to certain level of success, but not as widely accepted or practically amenable to replacing conventional antibodies.

Did you know that camel, llama, and shark naturally produce single heavy chain antibodies that can function as 13-16kDa fragments (yes if you have read previous Allele Blogs http://allelebiotech.com/blogs/2009/08/camelid-antibodies/)? They can easily be produced in bacteria, used directly inside live cells via transgene, fused to other proteins as a fusion tag, linked to DNA oligos as a detection module, or immobilized on beads for pull down or co-IP. Currently, these antibodies need to be selected by display after obtaining immunized antibody libraries. There is generally no commercial service for creating custom camelid antibodies at this time due to patent and other issues. Existing products are available for jelly fish GFP and DsRed derived RFP fusions. Publications using such a limited number of camelid antibodies have been amazing so far—dozens in top journals within the last few months and after only a short period of time since product launch.

New Product of the Week 05-09-10 to 05-16-10: RFP-Trap for mCherry, mRFP1, mOrange, mPlum, and mRuby etc.

Promotion of the Week 05-09-10 to 05-16-10: Purchase our ThermoExp500 PCR Thermocycer for $4,650.00, and qualify for $200 off or for a $300 credit toward any other Allele Biotech product or service! http://www.allelebiotech.com/allele3/EQ.php

Original BioTechniques Article http://www.biotechniques.com/news/biotechniquesNews/biotechniques-257771.html?utm_source=BioTechniques+Newsletters+%2526+e-Alerts&utm_campaign=b94f127de0-Methods+Newsletter&utm_medium=email

Tags: Alpaca, camelid antibodies, Chromotek, Fab, GFP-Trap, Hc antibodies, mCherry, mOrange, mPlum, mRFP1, mRuby, nAbs, RFP-Trap, scFv, single domain antibodies, VHH, VHH antibodies

Expanding the Camelid Antibody Product Line

While Chromotek GFP-Trap resin has become one of the best sellers from the Allele Biotech’ Camelid Antibody (VHH antibody) product line, more products have been added that will prove to be great tools for GFP-related research.

GFP is a powerful tool to study protein localization and dynamics in living cells. However, the photo stability and the quantum efficiency of GFP are not sufficient for Super-Resolution Microscopy (e.g. 3D-SIM or STED) of fixed samples from cells expressing GFP-fusion proteins to visualize specific structures. Furthermore, many cell biological methods such as HCl treatment for BrdU-detection, the EdU-Click-iT™ treatment or heat denaturation for FISH lead to disruption of GFP signal.

Now we offer our GFP-Trap Booster for reactivation, boosting and stabilization of GFP, suitable for acquiring strong and long lasting signals from GFP-fusion proteins. It is based on a specific GFP-binding protein as in GFP-Trap but coupled to the fluorescent dye ATTO 488 (from ATTO-TEC). For information, please read the product description of this week New Product of the Week: GFP-Trap booster, ABP-CM-GBOOSTR, http://www.allelebiotech.com/shopcart/index.php?c=221&sc=158

Promotion of the week: All mTFP1 and mWasabi fusion plasmids are 30% off for this week only

Preview of future new product: a similarly high quality product, the RFP-Trap that pulls down DsRed derived proteins including mRFP1, mCherry, mOrange, mPlum but also mRuby and RFP-tagged fusion proteins.

Tags: anti-GFP, Chromotek, GFP coIP, GFP-Trap, mCherry, mOrange, mPlum, mRFP1, mRuby RFP-tagged fusion proteins, nti-RFP, pulldown, RFP-Trap, VHH antibody