GFP as affinity tag for immunoprecipitation

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1 1/5 Preamble This document compares existing conventional antibodies against GFP in biochemical studies with the GFPTrap, a readytouse pulldown reagent derived from alpaca. What are the differences between the novel ChromoTek GFPTrap, which is based on a nanobody derived from alpaca, and conventional antigfp antibodies (figure 1)? Figure 1 Introduction Today, a considerable number of life science laboratories apply fluorescent proteins in cell biology to study protein localization, interaction and dynamics. Fluorescent proteins have redefined fluorescence microscopy because they are nontoxic and are less harmful when illuminated in live cells as compared to chemical fluorescent dyes. However, data generated from microscopic studies require complementary technologies to obtain a more comprehensive set of information. Additional aspects including posttranslational modifications, DNA binding, enzymatic activity, and/or complex formation respectively proteinprotein interaction are investigated. Mass spectrometry, chromatin immunoprecipitation (ChIP), coimmunoprecipitation and/or affinity purification methods are used to complement microscopic measurements. A common challenge for the above listed biochemical applications is the absence of validated antibodies against the protein of interest. Consequently, peptide and protein tags

2 2/5 like Myc, FLAG, HA, MBP and GST among others may be recombinantly fused to the protein of interest in order to utilize commercially available antibodies. Researchers now have the opportunity to apply fluorescent proteins as tags for those biochemical studies rather than recloning the genes coding for their protein of interest into vectors with traditional epitope tags. This is due to the availability of high quality immobilized GFPnanobody ChromoTek GFPTrap. GFPTrap vs. GFPantibody: workflow comparison GFPTrap : As shown in figure 2, after addition of cell lysate to the ChromoTek GFPTrap the immunoprecipitation of the GFPtagged protein takes place within a short incubation time; the entire workflow is typically completed within one hour because of high sensitivity, fast binding and extreme low dissociation constant. Conventional GFPantibody: In contrast, when adding cell lysate to traditional antibodies against GFP, the incubation may take up to 12 hours or overnight. Plus, in a second step, the GFP antibody complex needs to be coupled to protein A/G beads, which takes another 12 hours. This makes the entire workflow to last typically more than 3 hours or (much) longer. Figure 2: Workflow comparison between immunoprecipitations with alpaca Nanobody derived GFPTrap (left) and with conventional antigfp IgG antibodies (right). The high specificity and affinity allow for considerable shorter incubation times.

3 3/5 Immunoprecipitation results Input (I), nonbound (FT) and bound (B) fractions processed for IP with GFPTrap (Figure 3, left) and antigfp antibodies coupled to Protein A/G (Figure 3, right) were separated by SDSPAGE followed by Coomassie staining and Western Blotting. GFP has been purified using both approaches. The bound fraction of the GFPTrap shows only the purified GFPfusion protein of interest. In contrast, the bound fraction of the conventional GFP antibodies contains two additional strong bands besides the purified protein of interest. These bandsat approximately 50 kda and 25 kdaare contaminating heavy and light chains from the conventional antigfp antibody used for the immunoprecipitation. This may be a serious problem if you want to detect proteins of similar size. Figure 3: Immunoprecipitation of GFP from cell extracts. Protein extracts of GFPproducing HEK 293T cells were subjected to immunoprecipitation with GFPTrap or monoclonal antigfp antibody. Input (I), flowthrough (FT), and bound fractions (B) were separated by SDSPAGE and visualized by Coomassie Blue staining (top) or by immunoblot analysis (bottom). Precipitated GFP (green ellipse), denatured heavy (brown cartoon) and light chains (beige cartoon) of the IgG are marked by arrows.

4 4/5 Immunoprecipitation discussion: One challenge of the postgenomic era is the seamless integration of genetic, biochemical, and cell biological data. Here, we not only have demonstrated how the fluorescent protein GFP can be efficiently used as an affinity tag for biochemical studies of fusion proteins, but have also presented additional performance benefits: GFPTrap GFP antibody Ready to use Additional incubation step for binding of antibodies to protein A/G beads No heavy & light antibody chains in your downstream application Contaminating heavy & light antibody chains may appear in your downstream application Very high binding affinity Low binding affinity Harsh wash conditions may be applied for selective proteinprotein interactions Limited buffer/reagent compatibility of IgG Short incubation times of 530 minutes Considerable incubation times of twice minimum 1 hour; this may interfere with low affinity interaction partners if you perform CoIP High reproducibility due to recombinant expression with very low batch to batch variation Reproducibility can be high dependent on monoclonal but polyclonal can be different Not suitable as detection antibody in Western Blot Can generally be used as detection antibody in Western Blot Technology: GFPTrap Conventional antibodies are powerful tools in life science research. However, the large and complex structuretwo heavy and two light chainscan be troublesome in certain applications. Camelidae (alpacas, llamas, camels and dromedaries) possess a second type of antibody called heavy chain antibodies (hcabs). HcAbs are devoid of light chains and bind their antigen via a single variable domain (V H H), also known as a nanobody. These V H H domains have excellent binding properties and can be produced at constant high quality without batchtobatch variations. Coupled to an immobilizing matrix like agarose beads, V H Hs are superior tools for immunoprecipitations. Figure 4: GFPTrap : antigfp V HH coupled to agarose beads

5 5/5 Further reading: more than 700 publications Many researchers have successfully used the GFPTrap for a number of high impact publications that ChromoTek collects in a webbased database. We are frequently updating our database; currently there are more than 700 publications for the GFPTrap. You can filter your selection by organism, and type of experiment at our website to find relevant publications relevant for your studies. You can also find a section on frequently asked questions at ChromoTek, GFPTrap are registered trademarks of ChromoTek GmbH, Martinsried, Germany.