Anti-GFP antibody choices

A variety of anti-GFP antibodies are now provided to fluorescent protein users. In addition to the monoclonal anti-GFP antibody that has been introduced together with the GFP-Trap product group, Allele Biotech also has a strong anti-GFP polyclonal product, and a new rat anti-GFP monoclonal antibody. The availability of these different species specificity should allow users to have options in staining, detection, co-IP, double-labeling, etc.

New Product of the Week 04-05-10 to 04-11-10: Rat anti-AvGFP antibody, ACT-CM-MRGFP10

Promotion of the Week 04-05-10 to 04-11-10: Buy any Allele Biotech/Orbigen’s polyclonal antibody, get another of equal or less value at 60% off (if you are not a fan or friend through Allele’s online social networks, the discount is 30%).

Tags: 3H9, anti-GFP, antibodies, camelid antibodies, Chromotek, GFP booster, GFP detection, monoclonal, polyclonal, rat monoclonal

Using 2A “self-cleaving” peptide in bicistronic mammalian expression

Multiple promoters or internal ribosomal entry sites (IRES) have been used for the production of multiple proteins from the same vector. Potential drawbacks with multiple promoters on viral vectors include unstable genome and interference between promoters. IRES is a relatively large sequence that can cause problems in virus packaging, especially for viruses with very limited genome size such as AAV. In addition, it is required that the start of the second ORF is fairly close to the IRES, adding difficulties to cloning.

2A or 2A-like peptide (collectively called 2A peptide here) is used by several families of viruses, the best known foot-and-mouth disease virus of the Picornaviridae family, for producing multiple polypeptides. Although called a “self-cleaving” peptide or protease site, the mechanism by the 2A sequence for generating two proteins from one transcript is by ribosome skipping–a normal peptide bond is impaired at 2A, resulting in two discontinuous protein fragments from one translation even.

The 2A-based bicistronic expression has been used for several years, but recently gained much more popularity due to its successful use in iPSC generation that required 2 to 4 factors working in concert. Even expression of all factors can be achieved when 2A peptides are used for multiple protein production, due to near 100% efficiency of the 2A “cleavage” at each site, and no interference between multiple 2A sites. Early work used a 36 amino acid sequence as 2A peptide, which was later reduced to about half that size from mutation and screening. Commercial vectors utilizing 2A for co-expression of cDNA and fluorescent protein and/or drug resistance genes have not been available until now. Allele Biotech has introduced a number of such plasmids, establishing another First-to-the-market as it has done many times previously in its 10 year history.

New product of the week 03-29-10 to 04-04-10:

Alleleustrious pmTFP1-2A Bicistronic mammalian expression vector, ABP-FP-T2A10, $399, http://www.allelebiotech.com/shopcart/index.php?c=215&sc=34

Promotion of the week 03-29-10 to 04-04-10:

Buy any GFP-Trap beads or kits, get polyclonal anti-GFP (ABP-PAB-PAGFP10) at half size for FREE!  ***GFP-Trap has been replaced with GFP-nAb, an improved version for the same purposes in the form of agarose beads, magnetic beads, polyacrylamide beads, spin column kits, etc.  Come and take a look at the most comprehensive list of nearly all FPs before purchasing.

Tags: 2A peptide, anti-GFP, bicistronic, drug resistance, GFP-Trap, iPS, iPSCs, IRES, polycistronic, ribosome skip, self-cleaving, transcription, translation

Immunoprecipitation Tags

Immunoprecipitation is a process of isolating a protein as an antigen by using antibodies against it. It is a powerful tool for studying proteins in biological samples and, in case of Co-IP (meaning immunoprecipitation of complexes containing a known antigen), for analyzing protein-protein interactions. Similar technologies such as chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP), or crosslinked and iImmunoprecipitation of RNA-protein complexes (CLIP) aid analysis of protein-DNA or protein-RNA interactions.

The major obstacle for achieving effective immunoprecipitation is the difficulty of finding usable antibodies against a target of interest. A common practice is to use tags that are fused to the C- or N-terminus of the target protein, thereby any validated, commercially available antibody can be used for co-IP in different experimental systems. However, caution must be exercised against potential interference of biological functions from the added tags. In general, one should choose tags that have been tested in many situations and proven non-interfering; still, each biological system is different. Independent validation or supporting data should be used when interpreting results from tag-based co-IP.

Tags are often selected based on high quality and commercially available antibodies. Most commonly used tags include: FLAG, Myc, HA, V5, T7, and His, which are quite small in size and in theory less likely to interfere. GST and GFP are in between 20-30kDa, but they are well documented to form self-contained and stable structures independent of their fusion partners and proved to not interfere in many cases. GST can bind to glutathione beads directly, therefore a top choice for pulldown experiments. GFP or other FPs as tags have the advantages of being also a visualization module to follow the protein both inside cells and during pulldown. However, previously available anti-GFP antibodies, either polyclonal or monoclonal, are not comparable to those against other tags, thereby limiting the use of GFP as fusion tag in pulldown experiments.

GFP-Trap, a recent addition to anti-tag antibodies, is an E. coli expressed, single domain fragment derived from camelid heavy chain antibodies (VHH antibodies) with much higher stability, specificity, and affinity, making GFP based pulldown quantitative. This recent advancement should make GFP in line to become the most suitable tags for many aforementioned precipitation experiments.

Tags: alpaca antibodies, and His, camelid, chromatin immunoprecipitation (ChIP), Chromotek, Co-IP, FLAG, GFP, GST, HA, immunoprecipitation, Myc, nangobody, nanobodies, or crosslinked and iImmunoprecipitation of RNA-protein complexes (CLIP), protein-DNA, protein-protein, protein-RNA, RNA immunoprecipitation (RIP), single domain, T7, V5, VHH, VHH antibody

Camelid Antibodies

When it was discovered that animals in the camel family produce antibodies with no light chains, the idea that a single-domain fragment can bind as well as a full 4-chain antibody formed a breakthrough. So far it has been a relatively less known one.

Smaller antibody fragments have been tested for therapeutic uses because classical IgG antibodies are too bulky to penetrate tissues well, and very expensive to produce. Different combinations of antigen-binding variable regions are used, e.g. scFv, Fab, diabody, all to some degree of success. In comparison, the N-terminal domain of camelid antibodies, termed VHH domain (nanobody, VHH antibody), represents a naturally evolved, only 13-15 kD in size, fully functional target binding fragment with many advantages.

The only other known species outside camelidae family that has heavy chain antibodies is particular cartilaginous fish, nurse shark. Although the arrangement of CDRs is somewhat different between the camel and shark heavy chain variable regions, they share many characteristics such as extremely high stability (maintaining functions after100 C heat and extreme pH treatment).

Accumulating reports have demonstrated the therapeutic potentials of camelid antibody-based fragments in treating cancer, neural diseases, even use in hair dandruff preventing shampoo. For basic research, the tiny antigen binders can be used as tools for quantitative pull down with unmatched efficiency, recognizing previously inaccessible enzyme cleft as antigens, and providing libraries for binding partner selection.

Allele Biotech has been working on display antibody selection from its early days through an NIH grant, and recently carried out an NIH/NCI contract for scFv yeast display.

Check out Allele’s current Camelid antibody products: http://www.allelebiotech.com/allele3/CM.php

Tags: alpaca antibodies, antibody fragment, camelid antibodies, Chromotek, GFP fusion, GFP-Trap, llama antibody, nAb: Camelid Antibodies, nanobodies, nanobody, nurse shark, pull down, shark antibodies, shark antibody, single domain, VHH, VHH antibody