Application Note Ubiquitination of GFP-tagged proteins

One step purification to test ubiquitination of GFP-tagged proteins

Background
Validation of candidate ubiquitinated proteins is often performed in vitro to avoid high-background fromendogenous ubiquitinated proteins in the cell. However, in vitro systems do not reflect the in vivo situation.
Critical questions whether those proteins would be modified in a more physiological setup or in different cell
types remain to be answered. Here fluorescent fusion proteins provide an efficient alternative toconventional in vitro systems based on recombinant protein, since they can be expressed within different
mammalian cell lines.
In our approach to analyze ubiquitination of individual candidate proteins in living cells we co-transfect theGFP-tagged protein with FLAG-tagged ubiquitin. Using the GFP-Trap, GFP-tagged proteins are efficiently
extracted from cellular extracts under non-denaturing conditions. Due to strong binding of the GFP-Trap incombination with covalent ubiquitination, highly specific washing steps under denaturing conditions (8MUrea and 1% SDS) are possible to remove unspecifically bound proteins. Samples can be further analyzed
by immunoblotting and mass spectrometry.

Materials and Methods
GFP-tagged candidate ubiquitin substrates were expressed in BG2 Drosophila neuronal cells in a 6-wellplate, together with FLAG-tagged ubiquitin. Transfected cells were lysed in 300 μl of lysis buffer (50 mM
Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% Triton, 1x Proteases Inhibitor cocktail (Roche AppliedScience), 0.7% N-ethylmaleimide). 15 µL of pre-equilibrated GFP-TrapA suspension was added to thecleared supernatant and incubated at RT for 150 min with gentle rolling. The beads were pelleted bycentrifugation at 2700xg for 2 min. The supernatant was removed and the beads were washed once withdilution buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 1x Proteases Inhibitor cocktail,0.7% N-ethylmaleimide) followed by three times washing with stringent washing buffer (8 M Urea, 1%SDS in 1x PBS) and once with 1% SDS in 1x PBS. Bound proteins were eluted in SDS-sample buffer(250 mM Tris-HCl pH 7.5, 40% glycerol, 4% SDS, 0.2% bromphenol blue) boiled at 95ºC for 10 min andloaded onto 4-15% Tris-Glycine gels. Immunoblot was performed and transferred to PVDF membrane
Application Note Ubiquitination of GFP-tagged proteins1

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Application Note Ubiquitination of GFP-tagged proteinsusing the iBlot system (Invitrogen). After antibody incubation, the membrane was developed using ECL kit(GE Healthcare) following the manufacturer’s instructions. The following antibodies were used:monoclonal mouse anti-GFP antibody; monoclonal mouse anti-Flag M2 HRP-conjugated antibody.

Results
Using GFP only expressing cells as a negative control, we tested for ubiquitination of 6 candidate proteinspreviously identified as being ubiquitinated in the developing brain of Drosophila (1). Immunoblot analysis
reveals that all six proteins displayed FLAG immunoreactivity above their corresponding unmodified band(Figure 1), as expected by addition of one or several ubiquitin moieties (each Ub-FLAG chain should add~8 kDa). No ubiquitin FLAG signal was detected on the free GFP control construct despite using a verylong exposure. The GFP antibody does not detect the ubiquitin modified forms of the GFP-fusion
proteins, due to their low abundance, but only the main unmodified fraction for each of the substrates.

Figure 1.
Ubiquitination assay for six candidate ubiquitin
substrate proteins in BG2 neuronal cell culture.
Mouse anti-GFP antibody was used for detecting thecaptured proteins (shown in green), and anti-FLAG
HRP-conjugatedantibodyfor

monitoring

theirubiquitinated fraction (shown in red). On the first lane,
GFP on its own displays no ubiquitination. Othertypes of ubiquitin-tagging could be used for improveddetection, for example biotin (2) as well as available
ubiquitin antibodies to detect specific chain linkage

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Application Note Ubiquitination of GFP-tagged proteins

Conclusion
We showed that the GFP-Trap can withstand extremely stringent washes (8M Urea and 1% SDS)
without losing its affinity to its antigen, a GFP-fusion protein. This facilitates the isolation andcharacterization of individual proteins which are subjected to posttranslational modifications, likeubiquitination. As this modification is covalently attached to its target it is not disrupted by the highly
denaturing buffers used herein. As modified protein samples are eluted from the beads by boiling withsample loading buffer, this approach is compatible with both immunoblotting and Mass spectrometry afterin-gel digestion. The strategy presented here based on the high affinity GFP-Trap in combination withstringent washing procedures can be expanded for other covalent posttranslational modification likesumoylation.

References/relevant literature(1) M. Franco, N. T. Seyfried, A. H. Brand, J. Peng and U. Mayor.

A novel strategy to isolate ubiquitin conjugates reveals wide role of ubiquitination during neural development .
Molecular & Cellular Proteomics (2011) 10(5):M110.002188.
(2) M. Min, U. Mayor and C. Lindon. Ubiquitination site preferences in APC/C substrates.
Open Biol. (2013) 3:130097; doi:10.1098/rsob.130097, 2046-2441
S.Y. Lee, J.Ramirez, M.Franco, B.Lectez, M. Gonzalez, R. Barrio and U. Mayor

Ube3a, the E3 ubiquitin ligase causing Angelman syndrome and linked to autism, regulates protein homeostasis
through the proteasomal shuttle Rpn10.
Cellular and Molecular Life Sciences (2013) doi:10.1007/s00018-013-1526-7

AuthorUgo Mayor
Catherine Lindon

Ordering Information
ProductQuantityCode

20 reactions

gta-20

250 reactions

gta-250

GFP-Trap _A kit

20 reactions

gtak-20

GFP antibody

100 l3h9

GFP-Trap _A
GFP-Trap _A

http://www.chromotek.com

Application Note Ubiquitination of GFP-tagged proteins3

Version 2014-02-11