We would like to thank all members of the Kerscher lab for their support, Nathalie Nguyen for critical reading of the manuscript, and Lidia Epp for sequencing. This work has been supported by the Commonwealth Research Commercialization fund MF16-034-LS to OK. Research support for W&#38;M students was provided by the Bailey-Huston Research fund, and Charles Center Honors Fellowships to RY and CH.

1. SUMO detection in fixed tissue culture cells using recombinant KmUTAG-FL SUMO-trapping protein
<ol>
	<li>Grow tissue culture cells of choice on 22 mm round cover slips in 6-well TC plates until 70%&#8211;80% confluent. Perform steps 1.2&#8211;1.8 in the 6-well plate.</li>
	<li>Wash cells briefly with 1 mL of DPBS (Dulbecco&#39;s phosphate-buffered saline)</li>
	<li>For fixation, prepare a fresh solution of 4% Paraformaldehyde (PFA) solution (diluted in DPBS). To fix cells, add 2 mL 4% PFA in DPBS to each well. Incubate the cells for 20 min at room temperature. NOTE: All steps using PFA should be performed in a laboratory safety hood and PFA must be disposed properly.</li>
	<li>Wash the fixed cells 3x in 1 mL DPBS while nutating the plate, 5 min each wash.</li>
	<li>To permeabilize cells, incubate for 15 min with 0.1% Triton X-100 in DPBS</li>
	<li>Wash the cells 3x in 1 mL DPBS while nutating the plate, 5 min each wash</li>
	<li>Incubate the cells with 500 &#181;L of 0.1 M Glycine-HCL (pH 2.0) for 10 s, then neutralize pH immediately with 500 &#181;L of 10x SUMO Protease Buffer (SPB).</li>
	<li>Wash the cells 3x in 1 mL DPBS while nutating plate, 5 min each wash</li>
	<li>Remove the coverslips from the well and place them in a humidity chamber. Then proceed with incubations on the coverslip as follows:
	<ol>
		<li>For UTAG-fl staining only: mix 1 &#181;g UTAG-fl with 100 &#181;L of 1x SPB containing 5 mM TCEP in a tube, pipette the mix onto the cells on the coverslip, and incubate at room temperature for 1 h in the humidity chamber.</li>
		<li>Optionally, for UTAG-FL and anti SUMO1 antibody co-staining, proceed with the following:
		<ol>
			<li>Mix in a tube 1 &#181;g UTAG-FL and 0.5 &#181;L SUMO2/3 8A2 (obtained for Developmental Studies Hybridoma Bank9) with 100 &#181;L of blocking buffer, pipette the mix onto the coverslip, and incubate in room temperature for 1 h.</li>
			<li>Wash cells on the coverslip 3 times with 200 &#181;L DPBS, 5 min each wash.</li>
			<li>Mix in a tube 0.5 &#181;L anti-mouse Alexa Fluor 488 conjugated antibody with 100 &#181;L Blocking buffer, pipette the mix onto the coverslip, and incubate in room temperature for 1 h.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>To wash coverslips, pipette 200 &#181;L DPBS on each coverslip and leave in place for 10 min. Repeat the wash 2 more times.</li>
	<li>Remove coverslip from the last wash and invert it onto a pre-cleaned microscopy slide with a drop of mounting medium (see Table of Materials).</li>
	<li>Store overnight in a -20&#176;C freezer before viewing under the microscope. Visualize using the appropriate filter sets for DAPI (DNA), Texas Red (kmUTAG-fl), and Alexa Fluor 488 (if optional SUMO2/3 co-staining is performed).</li>
</ol>
2. SUMO detection in fixed nematode gonads using UTAG-fl
<ol>
	<li>Transfer adult hermaphrodites to an 8 &#181;L droplet of egg buffer14 on a plus-charged slide that has been coated with poly-L-lysine. Release gonads from the worms using 27.5 G needles. Proceed with either antibody labeling or UTAG-fl labeling.</li>
	<li>For antibody labeling samples, proceed as follows:
	<ol>
		<li>Freeze-crack samples in liquid nitrogen and then fix overnight in -20 &#176;C methanol in a Coplin jar.</li>
		<li>Also in Coplin jars, wash slides for 3 times in 1x PBS, then block for 20 min in PBS containing 0.5% BSA and 0.1% Tween 20.</li>
		<li>Add 30 &#181;L of anti-SUMO 6F2 antibody (1:10) to each slide covering the specimens. Incubate overnight in a humidity chamber at 4 &#176;C. 
		NOTE: SUMO 6F2 was obtained from the Developmental Studies Hybridoma Bank15.</li>
		<li>In Coplin jars, wash slides for 2 min in 1x PBS, then cover and incubate specimens with 30 &#181;L of DyLight 488 goat-anti mouse secondary antibody (1:200) for 1.5 hours in a humidity chamber at room temperature.</li>
		<li>In Coplin jars, wash slides for 2 min in 1x PBS, perform a quick dip in dH20, and then mount the slides with mounting medium (see Table of Materials).</li>
	</ol>
	</li>
	<li>For KmUTAG-fl labeling, proceed with the following:
	<ol>
		<li>To fix cells, add 1 volume of 8% PF to samples for a final concentration of 4% PF. Fix for 10 min in a humidity chamber and then quench the reaction by transferring slides to a Coplin jar containing 1x PBS with 0.1 M glycine for at least 5 min.</li>
		<li>In Coplin jars, wash cells for 5 min in 1x PBS, then permeabilize the samples in 1x PBS containing 0.1% Triton-X for 10 min.</li>
		<li>Wash in a Coplin jar for at least 5 min in 1x PBS, then add 200 &#181;L of 0.1 M Glycine-HCl (pH = 2.0) to the samples on the slide for 10 seconds. Immediately add 200 &#181;L of 10x SPB to neutralize the pH.</li>
		<li>Wash the slide in 1x PBS in a Coplin jar for 5 min.</li>
		<li>Remove slide from wash and pipette 100 &#181;L of 1x SPB + 5mM TCEP containing 2 &#181;g of UTAG-fl to the nematodes on the slides, and incubate in humidity chamber for 1 h without rocking.</li>
		<li>Return the slide to the Coplin jar and wash for 15 min in 1x PBS</li>
		<li>Remove the slide from the wash, use a laboratory wipe to dry around the sample, and then mount the slides with 5 &#181;L of mounting medium (see Table of Materials). Store the slides at 4 &#176;C. Visualize using the appropriate filter sets for DAPI (DNA), Texas Red (kmUTAG-fl), and DyLight 488 (SUMO2/3).</li>
	</ol>
	</li>
</ol>

<ol><li>Kerscher, O., Felberbaum, R., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modification+of+proteins+by+ubiquitin+and+ubiquitin-like+proteins%5BTitle%5D)+AND+%22Cell+and+Developmental+Biology%22%5BJournal%5D)">Modification of proteins by ubiquitin and ubiquitin-like proteins.</a> Cell and Developmental Biology. 22, 159-180 (2006).</li>
<li>Kerscher, O. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKerscher%2C+O+%22SUMOylation%22">SUMOylation</a>. , John Wiley & Sons, Ltd. Chichester. 1-11 (2016).</li>
<li>Hay, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SUMO%3A+a+history+of+modification%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">SUMO: a history of modification.</a> Molecular Cell. 18 (1), 1-12 (2005).</li>
<li>Fu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disruption+of+SUMO-specific+protease+2+induces+mitochondria+mediated+neurodegeneration%5BTitle%5D)+AND+%22PLoS+Genetics%22%5BJournal%5D)">Disruption of SUMO-specific protease 2 induces mitochondria mediated neurodegeneration.</a> PLoS Genetics. 10 (10), e1004579-e1004579 (2014).</li>
<li>Zhang, H., Kuai, X., Ji, Z., Li, Z., Shi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Over-expression+of+small+ubiquitin-related+modifier-1+and+sumoylated+p53+in+colon+cancer%5BTitle%5D)+AND+%22Cell+biochemistry+and+biophysics%22%5BJournal%5D)">Over-expression of small ubiquitin-related modifier-1 and sumoylated p53 in colon cancer.</a> Cell biochemistry and biophysics. 67 (3), 1081-1087 (2013).</li>
<li>Wang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SUMO-specific+protease+1+promotes+prostate+cancer+progression+and+metastasis%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">SUMO-specific protease 1 promotes prostate cancer progression and metastasis.</a> Oncogene. 32 (19), 2493-2498 (2013).</li>
<li>Karami, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+SUMO-Protease+SENP7S+Regulates+%CE%B2-catenin+Signaling+and+Mammary+Epithelial+Cell+Transformation%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Novel SUMO-Protease SENP7S Regulates β-catenin Signaling and Mammary Epithelial Cell Transformation.</a> Scientific Reports. 7 (1), 46477(2017).</li>
<li>Pelisch, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+SUMO-Dependent+Protein+Network+Regulates+Chromosome+Congression+during+Oocyte+Meiosis%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">A SUMO-Dependent Protein Network Regulates Chromosome Congression during Oocyte Meiosis.</a> Molecular Cell. 65 (1), 66-77 (2017).</li>
<li>Zhang, X. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SUMO-2%2F3+modification+and+binding+regulate+the+association+of+CENP-E+with+kinetochores+and+progression+through+mitosis%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">SUMO-2/3 modification and binding regulate the association of CENP-E with kinetochores and progression through mitosis.</a> Molecular Cell. 29 (6), 729-741 (2008).</li>
<li>Baker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reproducibility+crisis%3A+Blame+it+on+the+antibodies%5BTitle%5D)+AND+%22Nature+News%22%5BJournal%5D)">Reproducibility crisis: Blame it on the antibodies.</a> Nature News. 521 (7552), 274-276 (2015).</li>
<li>Wang, Z., Prelich, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quality+control+of+a+transcriptional+regulator+by+SUMO-targeted+degradation%5BTitle%5D)+AND+%22Molecular+and+Cellular+Biology%22%5BJournal%5D)">Quality control of a transcriptional regulator by SUMO-targeted degradation.</a> Molecular and Cellular Biology. 29 (7), 1694-1706 (2009).</li>
<li>Li, S. J., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+protease+required+for+cell-cycle+progression+in+yeast%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A new protease required for cell-cycle progression in yeast.</a> Nature. 398 (6724), 246-251 (1999).</li>
<li>Peek, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SUMO+targeting+of+a+stress-tolerant+Ulp1+SUMO+protease%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">SUMO targeting of a stress-tolerant Ulp1 SUMO protease.</a> PLoS ONE. 13 (1), e0191391(2018).</li>
<li>Edgar, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chapter+13+Blastomere+Culture+and+Analysis%5BTitle%5D)+AND+%22Methods+in+Cell+Biology%22%5BJournal%5D)">Chapter 13 Blastomere Culture and Analysis.</a> Methods in Cell Biology. 48, 303-321 (1995).</li>
<li>Pelisch, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+SUMO+modification+regulates+mitotic+chromosome+assembly+and+cell+cycle+progression+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Dynamic SUMO modification regulates mitotic chromosome assembly and cell cycle progression in Caenorhabditis elegans.</a> Nature Communications. 5 (1), 769(2014).</li>
<li>Dillingham, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescent+single-stranded+DNA+binding+protein+as+a+probe+for+sensitive%2C+real-time+assays+of+helicase+activity%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">Fluorescent single-stranded DNA binding protein as a probe for sensitive, real-time assays of helicase activity.</a> Biophysical Journal. 95 (7), 3330-3339 (2008).</li>
<li>Ruckman, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((2%27-Fluoropyrimidine+RNA-based+aptamers+to+the+165-amino+acid+form+of+vascular+endothelial+growth+factor+%28VEGF165%29.+Inhibition+of+receptor+binding+and+VEGF-induced+vascular+permeability+through+interactions+requiring+the+exon+7-encoded+domain%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">2'-Fluoropyrimidine RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165). Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain.</a> The Journal of Biological Chemistry. 273 (32), 20556-20567 (1998).</li>
<li>Hughes, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+specific+inhibitors+of+SUMO-1-+and+SUMO-2%2F3-mediated+protein-protein+interactions+using+Affimer+%28Adhiron%29+technology%5BTitle%5D)+AND+%22Science+Signaling%22%5BJournal%5D)">Generation of specific inhibitors of SUMO-1- and SUMO-2/3-mediated protein-protein interactions using Affimer (Adhiron) technology.</a> Science Signaling. 10 (505), eaaj2005(2017).</li>
<li>Lopata, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Affimer+proteins+for+F-actin%3A+novel+affinity+reagents+that+label+F-actin+in+live+and+fixed+cells%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells.</a> Scientific Reports. 8 (1), 6572(2018).</li>
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</ol>

Recombinant kmUTAG reagents are provided to the research community via Kerafast.com.

Here we introduce the use of kmUTAG-fl, a recombinant protein, for functional studies of SUMO in fixed mammalian cells and dissected nematode gonads. KmUTAG-fl is a stress-tolerant pan-SUMO specific reagent that recognizes and traps native SUMO-conjugated proteins and SUMO chains. Since SUMO&#39;s tertiary structure is highly conserved it is very likely that SUMO variants from additional model and non-model systems can be analyzed with the kmUTAG-fl reagent. As such, KmUTAG-fl may represent a useful alternative or second...

The purpose of this method is to facilitate the study and analysis of SUMO-conjugated proteins using the recombinant SUMO-trapping UTAG (Ulp domain Tag) protein. As detailed below, UTAG can be used in lieu of other reagents and approaches to purify, detect, and visualize SUMO-modified proteins. Depending on growth conditions, cells may contain hundreds or thousands of proteins that are modified with SUMO or SUMO chains (for review see Kerscher et al. 20061 and Kerscher 20162). This represents a considerable difficulty for the functional analyses of specific SUMO-modified proteins, especially since only a fraction of a particular sumoylation target is actually modified3. In addition to their roles in essential cellular processes such as transcriptional regulation, protein homeostasis, the response to cellular stress, and chromatin remodeling during mitosis and meiosis; it has now become sufficiently clear that SUMO, SUMO-modified proteins, and SUMO pathway components also have potential as biomarkers for pathologies such as cancer and neurodegenerative disorders4,5,6,7. This underscores the need for robust, reliable, and readily available tools and innovative approaches for the detection and functional analysis of SUMO-modified proteins in a variety of cells and samples.
In many systems, SUMO-specific antibodies are the reagents of choice for the detection, isolation and functional analyses of SUMO-modified proteins8,9. However, some commercially available SUMO-specific antibodies are expensive, limited in quantity or availability, prone to exhibit wildly variable affinities and cross-reactivity, and in some instances lack reproducibility10. One alternative approach is the expression of epitope-tagged SUMO in transformed cells and organisms, but linking epitope tags to SUMO may artificially lower its conjugation to protein targets11. Additionally, epitopes are not useful when untransformed cells or tissues are evaluated.
Ulp1 is a conserved SUMO protease from S. cerevisiae that both processes the SUMO precursor and desumoylates SUMO-conjugated proteins12. We developed the UTAG reagent based on the serendipitous observation that a mutation of the catalytic Cysteine (C580S) in Ulp1&#39;s SUMO processing Ulp domain (UD) not only prevents SUMO cleavage but also traps SUMO-conjugated proteins with high avidity12. For simplicity, we referred to this carboxy-terminal SUMO-trapping Ulp1(C580S) fragment as UTAG (short for UD TAG). UTAG is a recombinant pan-SUMO trapping protein that represents a useful alternative to anti-SUMO antibodies used for the isolation and detection of SUMO-modified proteins. Importantly, it specifically recognizes natively-folded, conjugated SUMO and not just one or several epitopes on SUMO. To improve both the protein stability and SUMO binding strength of UTAG, we generated a variant of UTAG from the stress-tolerant yeast Kluyveromyces marxianus (Km). KmUTAG tightly binds SUMO-conjugates with nanomolar affinity13. Additionally, kmUTAG is resistant to elevated temperatures (42 &#176;C), reducing agents (5 mM TCEP - Tris(2-carboxyethyl)phosphine hydrochloride), denaturants (up to 2 M Urea), oxidizing agents (0.6% hydrogen peroxide), and non-ionic detergents. This stress tolerance is beneficial during harsh purification condition and prolonged incubation times, ensuring its stability and SUMO-trapping activity. Not surprisingly, however, KmUTAG&#39;s SUMO-trapping activity is incompatible with cysteine-modifying reagents, ionic detergents and fully denatured protein extracts. The remarkable affinity and properties of KmUTAG indicate that this reagent may become part of the standard repertoire for the study of sumoylated proteins in multiple species.
Here we provide a simple method to detect SUMO-modified proteins in mammalian cells and nematodes using a recombinant fluorescent mCherry-KmUTAG fusion protein (kmUTAG-fl).

<table><tbody><tr><td>16% Paraformaldehyde (formaldehyde) aqueous solution</td><td>Electron Microscopy Sciences</td><td>30525-89-4</td><td></td></tr><tr><td>6-Well Cell Culture Plates</td><td>Genesee Scientific/Olympus Plastics</td><td>25-105</td><td></td></tr><tr><td>Alexa Fluor 488 AffiniPure Goat Anti-Mouse IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>115-545-003</td><td>Used as a secondary antibody for mouse monoclonal antibody</td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Fisher Scientific</td><td>Gibco 14190144</td><td></td></tr><tr><td>Dylight 488 conjugated AffiniPure Goat Anti-Moue IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>115-485-146</td><td>Used as a secondary antibody for mouse monoclonal antibody</td></tr><tr><td>Fisherbrand Coverglass for Growth Cover Glasses</td><td>Fisherbrand</td><td>12545101</td><td></td></tr><tr><td>FLUORO-GEL II with DAPI</td><td>Electron Microscopy Sciences</td><td>50-246-93)</td><td>Mounting media in step 1.11</td></tr><tr><td>FLUORO-GEL with DABCO</td><td>Electron Microscopy Sciences</td><td>17985-02</td><td>With DAPI added to 1 &amp;micro;g/mL; mounting media in step 2.2.5</td></tr><tr><td>Glycine-HCl</td><td>Fisher BioReagents</td><td>BP3815</td><td></td></tr><tr><td>Glycine-HCl</td><td>ACROS Organics</td><td>6000-43-7</td><td></td></tr><tr><td>KmUTAG-fl</td><td>Kerafast</td><td></td><td>KmUTAG reagents are available on Kerafast.com</td></tr><tr><td>Oneblock Western-CL blocking buffer</td><td>Prometheus</td><td>20-313</td><td></td></tr><tr><td>PBS, Phosphate Buffered Saline, 10X Solution</td><td>Fisher BioReagents</td><td>BP3994</td><td></td></tr><tr><td>PNT2 cell line</td><td>Sigma-Aldrich</td><td>95012613</td><td>Normal prostate epithelium immortalized with SV40.</td></tr><tr><td>pSPOT1</td><td>(ChromoTek GmbH)</td><td>ev-1</td><td>https://www.chromotek.com/fileadmin/user_upload/pdfs/Datasheets/pSpot1_v1.pdf</td></tr><tr><td>SUMO 6F2</td><td>DSHB</td><td>SUMO 6F2</td><td>SUMO 6F2 was deposited to the DSHB by Pelisch, F. / Hay, R.T. (DSHB Hybridoma Product SUMO 6F2)</td></tr><tr><td>SUMO protease buffer [10x]</td><td></td><td></td><td>500 mM Tris-HCl, pH 8.0, 2% NP-40, 1.5 M NaCl</td></tr><tr><td>SUMO-2 Antibody 8A2</td><td>DSHB</td><td>SUMO-2 8A2</td><td>SUMO-2 8A2 was deposited to the DSHB by Matunis, M. (DSHB Hybridoma Product SUMO-2 8A2)</td></tr><tr><td>TCEP-HCL</td><td>GoldBio</td><td>51805-45-9</td><td>Used as a reducing agent at a concentration of 5mM</td></tr><tr><td>Triton X-100</td><td>Fisher BioReagents</td><td>9002-93-1</td><td>Used for permeablization at 0.1% in DPBS/PBS(for worms)</td></tr></tbody></table>

localization of sumo-modified proteins using fluorescent sumo-trapping proteins

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. This method utilizes a recombinant modified SUMO-trapping protein fragment, kmUTAG, derived from the Ulp1 SUMO protease of the stress-tolerant budding yeast Kluyveromyces marxianus. We have adapted the properties of the kmUTAG for the purpose of studying sumoylation in a variety of model systems without the use of antibodies. For the study of SUMO, KmUTAG has several advantages when compared to antibody-based approaches. This stress-tolerant SUMO-trapping reagent is produced recombinantly, it recognizes native SUMO isoforms from many species, and unlike commercially available antibodies it shows reduced affinity for free, unconjugated SUMO. Representative results shown here include the localization of SUMO conjugates in mammalian tissue culture cells and nematode oocytes.

KmUTAG-fl is a recombinant, mCherry-tagged SUMO-trapping protein. To produce kmUTAG-fl, we cloned a codon-optimized mCherry-kmUTAG into the pSPOT1 bacterial overexpression plasmid (Figure 1). After induction, the kmUTAG-fl protein was purified on Spot-TRAP, eluted, and frozen until further use. To ensure the SUMO-trapping activity of KmUTAG-fl, we confirmed binding to SUMO1-conjugated beads and precipitation of a SUMO-CAT fusion protein (data not shown, but s...

Watch this Scientific Journal Video about Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins at JoVE.com

Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins

SUMO is an essential and highly conserved, small ubiquitin-like modifier protein. In this protocol we are describing the use of a stress-tolerant recombinant SUMO-trapping protein (kmUTAG) to visualize native, untagged SUMO conjugates and their localization in a variety of cell types.

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. ...

localization-sumo-modified-proteins-using-fluorescent-sumo-trapping

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Biochemistry

8.4K Views.  College of William & Mary. De betekenis van dit protocol is het gebruik van een nieuwe fluorescerende SUMO-trapping UTAG eiwit voor de detectie van het eiwit modifier SUMO in verschillende eukaryotische cellen. Het belangrijkste voordeel van deze techniek is een eenvoudig protocol voor de antilichaamvrije detectie van SUMO in verschillende cellen, waaronder de weefselkweek van zoogdieren en nematodeklieren. SUMO speelt een belangrijke rol bij kanker en neurodegeneratieve aandoeningen, en dit onderstreept de noodzaak va...

Video: Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

SUMO-Binding Entities (SUBEs) as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in liver cancer, the modification by SUMO (Small Ubiquitin MOdifier) has been given the most attention. Isolation of endogenous SUMOylated proteins in vivo is challenging due to the presence of active SUMO-specific proteases. Initial studies of SUMOylation in vivo were based on the molecular detection of specific SUMOylated proteins (e.g., by western blot). However, in many cases, antibodies, generally made with non-modified recombinant protein, did not immunoprecipitate SUMOylated forms of the protein of interest.&#160;Nickel chromatography has been the other approach to study SUMOylation by capturing histidine-tagged versions of SUMO molecules. This approach is mainly used in cells stably expressing or transiently transfected with His-SUMO molecules. To overcome these limitations, SUMO-binding entities (SUBEs) were developed to isolate endogenous SUMOylated proteins. Herein, we describe all the steps required for the enrichment, isolation, and identification of SUMOylated substrates from human hepatoma cells and hepatic tissues from a liver cancer mouse model by using SUBEs. Firstly, we describe methods involved in the preparation and lysis of the human hepatoma cells and liver tumor tissue samples. Then, a thorough explanation of the preparation of SUBEs and controls is detailed along with the protocol for the protein pull-down assays. Finally, some examples are provided regarding the options available for the identification and characterization of the SUMOylated proteome, namely the use of western-blot analysis for the detection of a specific SUMOylated substrate from liver tumors or the use of proteomics by mass spectrometry for high-throughput characterization of the SUMOylated proteome and interactome in hepatoma cells.

SUMO-Binding Entities SUBEs as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Here, we present a protocol to enrich, isolate, identify, and characterize proteins modified by SUMO in vivo both from human hepatoma cells and liver tumors obtained from mouse models of hepatocellular carcinoma by using SUMO-binding entities (SUBEs).

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in ...

Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. Imaging studies are widely used to investigate their function and plasticity in physiological and pathological conditions. Because of their size and structure, localization studies of proteins require high-resolution imaging techniques. In this protocol, we describe a procedure to study in primary neurons the co-localization of target proteins with synaptic markers at a super-resolution level using structured illumination microscopy (SIM). SIM is a patterned-light illumination technique that doubles the spatial resolution of wide-field microscopy, reaching a detail of around 100 nm. The protocol indicates the required controls and settings for robust co-localization studies and an overview of the statistical methods to analyze the imaging data properly.

This protocol shows how to employ super-resolution microscopy to study protein co-localization in primary neuronal cultures.

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. ...

Neuroscience

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular function of many proteins. PTMs of kinetochore proteins that associate with centromeric DNA mediate faithful chromosome segregation to maintain genome stability. Biochemical approaches such as mass spectrometry and western blot analysis are most commonly used for identification of PTMs. Here, a protein purification method is described that allows the detection of both sumoylation and ubiquitination of the kinetochore proteins, Ndc10 and Ndc80, in Saccharomyces cerevisiae. A strain that expresses polyhistidine-Flag-tagged Smt3 (HF-Smt3) and Myc-tagged Ndc10 or Ndc80 was constructed and used for our studies. For detection of sumoylation, we devised a protocol to affinity purify His-tagged sumoylated proteins by using nickel beads and used western blot analysis with anti-Myc antibody to detect sumoylated Ndc10 and Ndc80. For detection of ubiquitination, we devised a protocol for immunoprecipitation of Myc-tagged proteins and used western blot analysis with anti-Ub antibody to show that Ndc10 and Ndc80 are ubiquitinated. Our results show that epitope tagged-protein of interest in the His-Flag tagged Smt3 strain facilitates the detection of multiple PTMs. Future studies should allow exploitation of this technique to identify and characterize protein interactions that are dependent on a specific PTM.

This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular ...

Biology

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, medial-Golgi, trans-Golgi and trans-Golgi network. Cellular functions of the Golgi are determined by the characteristic distribution of its resident proteins. The spatial resolution of conventional light microscopy is too low to resolve sub-Golgi structure or cisternae. Thus, the immuno-gold electron microscopy is a method of choice to localize a protein at the sub-Golgi level. However, the technique and instrument are beyond the capability of most cell biology labs. We describe here our recently developed super-resolution method called Golgi protein localization by imaging centers of mass (GLIM) to systematically and quantitatively localize a Golgi protein. GLIM is based on standard fluorescence labeling protocols and conventional wide-field or confocal microscopes. It involves the calibration of chromatic-shift aberration of the microscopic system, the image acquisition and the post-acquisition analysis. The sub-Golgi localization of a test protein is quantitatively expressed as the localization quotient. There are four main advantages of GLIM; it is rapid, based on conventional methods and tools, the localization result is quantitative, and it affords ~ 30 nm practical resolution along the Golgi axis. Here we describe the detailed protocol of GLIM to localize a test Golgi protein.

The precise localization of Golgi residents is essential for understanding the cellular functions of the Golgi. However, conventional optical microscopy is unable to resolve the sub-Golgi structure. Here we describe the protocol for a conventional microscopy based super-resolution method to quantitatively determine the sub-Golgi localization of a protein.

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, ...

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions. However, due to the complexity of the process of SUMOylation in vivo, it is more challenging to detect and analyze protein SUMOylation in cells, especially when under endogenous conditions. A recent study by the authors of this paper revealed that endogenous retinoblastoma (Rb) protein, a tumor suppressor that is vital to the negative regulation of the cell cycle progression, is specifically SUMOylated at the early G1 phase. This paper describes a protocol for the detection and analysis of Rb SUMOylation under both endogenous and exogenous conditions in human cells. This protocol is appropriate for the phenotypical and functional investigation of the SUMO-modification of Rb, as well as many other SUMO-targeted proteins, in human cells.

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, ...

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily through modulating protein-protein interactions. Similar to ubiquitin modification, SUMOylation is directed by a sequential enzyme cascade including E1-activating enzyme (SAE1/SAE2), E2-conjugation enzyme (Ubc9), and E3-ligase (i.e., PIAS family, RanBP2, and Pc2). However, different from ubiquitination, an E3 ligase is non-essential for the reaction but does provide precision and efficacy for SUMO conjugation. Proteins modified by SUMOylation can be identified by in vivo assay via immunoprecipitation with substrate-specific antibodies and immunoblotting with SUMO-specific antibodies. However, the demonstration of protein SUMO E3 ligase activity requires in vitro reconstitution of SUMOylation assays using purified enzymes, substrate, and SUMO proteins. Since in the in vitro reactions, usually SAE1/SAE2 and Ubc9, alone are sufficient for SUMO conjugation, enhancement of SUMOylation by a putative E3 ligase is not always easy to detect. Here, we describe a modified in vitro SUMOylation protocol that consistently identifies SUMO modification using an in vitro reconstituted system. A step-by-step protocol to purify catalytically active K-bZIP, a viral SUMO-2/3 E3 ligase, is also presented. The SUMOylation activities of the purified K-bZIP are shown on p53, a well-known target of SUMO. This protocol can not only be employed for elucidating novel SUMO E3 ligases, but also for revealing their SUMO paralog specificity.

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with ~15&#8211;27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of self-labeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondrial compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.

Here, we present a protocol for multi-color localization of single membrane proteins in organelles of live cells. To attach fluorophores, self-labeling proteins are used. Proteins, located in different membranes compartments of the same organelle, can be localized with a precision of ~18 nm.

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a ...

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.

We here introduce a procedure to measure protein oligomers and aggregation in cell lysate and live cells using fluorescence correlation spectroscopy.

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's ...

Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins

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