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How to plan an immunoprecipitation of your GFP-fusion
protein when using the ChromoTek GFP-Trap
Preamble
This document provides
practical information on how to apply the ChromoTek GFP-Trap for
immunoprecipitation.
Introduction
The ChromoTek GFP-Trap is based on a GFP-binding protein derived
from an Alpaca
single variable domain antibody, also called VHH or nanobody
(figure 1). The GFP-Trap has particular properties and provides
some advantages over traditional IgG antibodies when applied in
immunoprecipitations.
Planning of the experiment
There are
some experimental aspects that you should consider when planning
immunoprecipitation of your GFP-fusion protein of interest using
the ChromoTek GFP-Trap . Below, we guide you through each
step.
There are 3 matrices available:
CRAPome:
By nature, every matrix and binding molecule may nonspecifically
bind some proteins
resulting in protein background. Scientists have established the
internet-based database
CRAPome at www.crapome.org. This database stores and annotates
negative controls
generated by the proteomics research community. CRAPome helps to
determine the
background contaminants--for example, proteins that interact with
the solid-phase support,affinity reagent or epitope
tag.
Specificity - What fluorescent proteins are captured
by the GFP-Trap
The GFP-Trap specifically binds to
most of the common GFP derivatives:
The ChromoTek GFP-Trap
only binds properly folded active GFP. It is believed that this is
because that nanobody binds to a three-dimensional epitope of GFP.
The nanobody’s
elongated CDR3 (complementarity determining region 3) allows to
reach into clefts and
enzymatic centers of proteins, which are not accessible to
conventional antibodies but results in very strong binding and very
low dissociation constants of this GFP nanobody. Therefore that
anti-GFP nanobody is not suitable for protein detection in Western
Blots. For Western Blot detection of GFP (fusion proteins)
ChromoTek recommends the traditional antibody anti-GFP antibody 3H9
(rat monoclonal, see complementary products).
Controls What controls
should I conduct to validate the experimental data?
Below find some suggestions by application:
For
Immunoprecipitation (IP):
GFP-Trap for IP of GFP-fusions and a non-relevant Nano-Trap as
negative 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 a
cell lysate?
Lysis buffers:
A non-denaturing lysis buffer is suitable for Co-IP, because
proteins will remain in
their native conformation
The RIPA (Radio Immunoprecipitation Assay) buffer might denature
proteins or
disrupt protein complexes
Inhibitors:
Add protease inhibitors to prevent proteolysis!
Preserve posttranslational modifications of your protein and add
e.g. phosphatase
inhibitors!
Prevent degradation of your protein by keeping your samples on
ice!
Immunoprecipitation Binding of the
GFP-fusion
Since the GFP binding protein is covalently coupled to the beads’
surface, the GFP-Trap beads are ready-to-use and can be directly
added to the prepared lysate. The affinity ofGFP-nanobody is in the
picomolar range, therefore depletion of GFP-fusions can becompleted
within 5-30 minutes.
Buffer compatibility of the GFP-Trap for binding
and washing
The GFP-Trap is compatible with most wash buffers and stable under
harsh conditions
including:
Up to 1 M NaCl and 8 M Urea
Up to 0.2% SDS and 2% NP-40
Elution strategies
The elution of the bound GFP-fusion protein by a competitive
peptide, which replaces the
GFP-fusion protein doesn’t work. Also, the addition of chaotropic
compounds like urea don’telute the bond GFP-fusion protein as the
GFP-Trap works under denaturing conditions. We therefore recommend
to elute with:
SDS, e.g. SDS sample buffer, is a very effective way to elute the
bound GFP-tagged
protein. The elution results in denatured GFP-fusions.
0.2 M glycine pH 2.5
Alternatively you may elute with glycine at pH 2.5. It is
recommended to repeat this
elution step as the pH shift elution works incompletely. The
repetition will improve the
elution efficiency.
Very important: Don’t forget to neutralize proteins immediately
after elution!
As an
alternative to above elution options, a protease cleavage site
between GFP and the fusion protein can be introduced. This option
is recommended if less stable proteins have
been bound or if you want to enrich your native protein of
interest.
Furthermore, consider whether you really need to elute the bound protein of interest from the beads rather than conduct the downstream analysis “on-bead”:
Proteins can be digested when still coupled to the beads for
subsequent mass
spectrometry analysis.
Enzymatic activity assays can be performed when still coupled to
the beads if the
active center is not blocked.
Reproducibility
The GFP-Trap is a small, soluble and stable single polypeptide
chain that is recombinantly expressed in bacteria. This in
combination with quality control makes its production robust and
reproducible for reliable results.
Selected References to
introduce Nanobodies and their
applications:
Nanobodies as probes for protein dynamics in vitro and in
cells
Dmitriev, O.
Y., Lutsenko, S. and Muyldermans, S. in: Journal of Biological
Chemistry, 2015 jbc-R115.
Nanobody-based products as research and diagnostic
tools
De Meyer, T.,
Muyldermans,S. and Depicker, A. in: Trends in Biotechnology, 2014
May; 32 (5): 263-270;
Beneficial properties of single-domain antibody fragments
for application in immunoaffinity purification and immuno-perfusion
chromatography
Verheesen 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
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