How to plan an immunoprecipitation of your GFP-fusionprotein when using the ChromoTek GFP-Trap

PreambleThis document providespractical information on how to apply the ChromoTek GFP-Trap forimmunoprecipitation.

IntroductionThe ChromoTek GFP-Trap is based on a GFP-binding protein derivedfrom an Alpacasingle variable domain antibody, also called VHH or nanobody(figure 1). The GFP-Trap has particular properties and providessome advantages over traditional IgG antibodies when applied inimmunoprecipitations.

Planning of the experimentThere aresome experimental aspects that you should consider when planningimmunoprecipitation of your GFP-fusion protein of interest usingthe ChromoTek GFP-Trap . Below, we guide you through eachstep.

There are 3 matricesavailable:

Pre-clearing is an optional step to remove proteins or DNA whichbind non-specifically to the solid-phase support. It includes theincubation of the cell extract with plain beads (e.g.bindingcontrol agarose beads) before performing the actualimmunoprecipitation experiment with the GFP-Trap. After successfulpre-clearing, non-specific proteins or other components will not beco-purified with the protein of interest.

CRAPome:By nature, every matrix and binding molecule may nonspecificallybind some proteinsresulting in protein background. Scientists have established theinternet-based databaseCRAPome at www.crapome.org. This database stores and annotatesnegative controlsgenerated by the proteomics research community. CRAPome helps todetermine thebackground contaminants--for example, proteins that interact withthe solid-phase support,affinity reagent or epitopetag.

Specificity - What fluorescent proteins are capturedby the GFP-Trap The GFP-Trap specifically binds tomost of the common GFP derivatives:

The ChromoTek GFP-Trap only binds properly folded active GFP. It is believed that this isbecause that nanobody binds to a three-dimensional epitope of GFP.The nanobody’selongated CDR3 (complementarity determining region 3) allows toreach into clefts andenzymatic centers of proteins, which are not accessible toconventional antibodies but results in very strong binding and verylow dissociation constants of this GFP nanobody. Therefore thatanti-GFP nanobody is not suitable for protein detection in WesternBlots. For Western Blot detection of GFP (fusion proteins)ChromoTek recommends the traditional antibody anti-GFP antibody 3H9(rat monoclonal, see complementary products).

Controls What controlsshould I conduct to validate the experimental data?Below find some suggestions by application:

ForImmunoprecipitation (IP): GFP-Trap for IP of GFP-fusions and a non-relevant Nano-Trap asnegative control,e.g. Myc-Trap , GST-Trap or MBP-Trap

For Co-Immunoprecipitation(Co-IP) of protein complex AB:

Cell Lysis What to consider when preparing acell lysate?Lysis buffers: A non-denaturing lysis buffer is suitable for Co-IP, becauseproteins will remain intheir native conformation The RIPA (Radio Immunoprecipitation Assay) buffer might denatureproteins ordisrupt protein complexesInhibitors: Add protease inhibitors to prevent proteolysis! Preserve posttranslational modifications of your protein and adde.g. phosphataseinhibitors! Prevent degradation of your protein by keeping your samples onice!

Immunoprecipitation Binding of theGFP-fusionSince the GFP binding protein is covalently coupled to the beads’surface, the GFP-Trap beads are ready-to-use and can be directlyadded to the prepared lysate. The affinity ofGFP-nanobody is in thepicomolar range, therefore depletion of GFP-fusions can becompletedwithin 5-30 minutes.

Buffer compatibility of the GFP-Trap for bindingand washingThe GFP-Trap is compatible with most wash buffers and stable underharsh conditionsincluding: Up to 1 M NaCl and 8 M Urea Up to 0.2% SDS and 2% NP-40

Elution strategiesThe elution of the bound GFP-fusion protein by a competitivepeptide, which replaces theGFP-fusion protein doesn’t work. Also, the addition of chaotropiccompounds like urea don’telute the bond GFP-fusion protein as theGFP-Trap works under denaturing conditions. We therefore recommendto elute with:

SDS, e.g. SDS sample buffer, is a very effective way to elute thebound GFP-taggedprotein. The elution results in denatured GFP-fusions. 0.2 M glycine pH 2.5Alternatively you may elute with glycine at pH 2.5. It isrecommended to repeat thiselution step as the pH shift elution works incompletely. Therepetition will improve theelution efficiency.

Very important: Don’t forget to neutralize proteins immediatelyafter elution!

As analternative to above elution options, a protease cleavage sitebetween GFP and the fusion protein can be introduced. This optionis recommended if less stable proteins havebeen bound or if you want to enrich your native protein ofinterest.

Furthermore, consider whether you really need to elute thebound protein of interest from the beads rather than conduct thedownstream analysis \"on-bead”:

Proteins can be digested when still coupled to the beads forsubsequent massspectrometry analysis. Enzymatic activity assays can be performed when still coupled tothe beads if theactive center is not blocked.

ReproducibilityThe GFP-Trap is a small, soluble and stable single polypeptidechain that is recombinantly expressed in bacteria. This incombination with quality control makes its production robust andreproducible for reliable results.

Selected References tointroduce Nanobodies and theirapplications:Nanobodies as probes for protein dynamics in vitro and incellsDmitriev, O.Y., Lutsenko, S. and Muyldermans, S. in: Journal of BiologicalChemistry, 2015 jbc-R115.

versatile nanotrap for biochemical and functional studies withfluorescent fusion proteinsRothbauer, U.,Zolghadr, K., Muyldermans, S., Schepers, A., Cardoso, M. C.,Leonhardt, H. in: Mol Cell Proteomics, 2008 Feb;7(2):282-9. Epub2007 Oct 21

Nanobody-based products as research and diagnostictoolsDe Meyer, T.,Muyldermans,S. and Depicker, A. in: Trends in Biotechnology, 2014May; 32 (5): 263-270;

Beneficial properties of single-domain antibody fragmentsfor application in immunoaffinity purification and immuno-perfusionchromatographyVerheesen P.,Ten Haaft M. R., Lindner N., Verrips C.T., de Haard J. J. W. in:Biochim. Biophys. Acta, 2003; 1624(1 3):21 28

highly specific gold nanoprobe for live-cell single-moleculeimagingLeduc, C., Si,S., Gautier, J., Soto-Ribeiro, M., Wehrle-Haller, B., Gautreau, A.,.Giannone, G., Cognet, L. Lounis, B. in:Nano letters, 2013: 13(4), 1489-1494.

Nanobody-based chromatin immunoprecipitation.Duc, T. N.,Hassanzadeh-Ghassabeh, G., Saerens, D., Peeters, E., Charlier, D., Muyldermans, S. in:Single Domain Antibodies: Methods and Protocols, 2013:491-505

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