We thank Diana Adjaoud for technical assistance. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (2015-2020), Genome Quebec (2016-2019) and Genome Canada (2016-2019) to E.B.A. E.B.A. is a senior scholar of the Fonds de la Recherche du Qu&#233;bec-Sant&#233; (FRQ-S). L.M and N.S.K. have a PhD scholarship from the FRQ-S. H.B had a PhD scholarship from the Ministry of Higher Education and from Scientific Research of Tunisia and the Cole Foundation.

1. GSH-agarose Affinity Purification of the GST-RING1B(1-159)-BMI1(1-109) E3 Ubiquitin Ligase Complex
<ol>
	<li>Use the pGEX6p2rbs-GST-RING1B (1-159 aa) - BMI1 (1 -109aa) bacteria expression construct to transform BL21 (DE3) bacteria (see Table of Materials)23. This construct allows the expression of murine RING1B domain 1-159 fused to BMI1 domain 1-109 with GST tag in pGEX-6P-2 backbone.</li>
	<li>Perform an overnight starter culture by inoculating RIL-bacteria expressing GST-RING1B (1-159 aa) - BMI1(1 -109 aa) in 20 mL of LB broth medium in presence of 100 &#181;g/mL ampicillin and 50 &#181;g/mL chloramphenicol. Incubate by shaking at 37 &#176;C overnight. On the next day, add the starter culture to a 1 L flask containing 500 mL of fresh LB broth medium (1/26 dilution) with ampicillin and incubate the culture in the shaker at 37 &#176;C for 2 to 4 h.</li>
	<li>During the incubation time, measure the OD at 600 nm with a spectrophotometer. When bacteria culture reaches 0.6 OD units at 600 nm, take a 1 mL aliquot in an 1,5 mL tube as the non-induced sample. Add 400 &#181;M of Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) to the 1 L culture to induce protein expression. Incubate bacteria at 25 &#176;C for 6 h to overnight.
	<ol>
		<li>Centrifuge the 1 mL aliquots at 14,000 x g for 30 s, discard the supernatant, resuspend the pellet in 200 &#956;L of Laemmli sample buffer, and keep for SDS-PAGE and Coomassie blue analysis of protein induction.</li>
	</ol>
	</li>
	<li>Transfer the induced bacteria culture from the flask into a clean centrifugation bottle and spin down at 3,500 x g for 15 min at 4 &#176;C. Discard the supernatant and resuspend the pellet with 40 mL of cold PBS and spin down at 3,500 x g for 15 min at 4 &#176;C.</li>
	<li>Discard the supernatant and freeze the pellet at -80 &#176;C or proceed to the next step. If the proteins are induced at the expected molecular weight, proceed to the next step.</li>
	<li>Resuspend the bacteria pellet in 25 mL of ice-cold lysis buffer A (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, 1% NP40, 1 mM PMSF, 0.5 mM DTT, and 1/100 anti-protease cocktail containing AEBSF, Aprotinin, Bestatin, E-64, Leupeptin, and Pepstatin A). Make sure that all the bacteria pellet is resuspended. PMSF and anti-protease cocktail must be freshly added to the lysis buffer, only at the time of bacteria lysis. 
	CAUTION: Always keep the sample on ice. 
	NOTE: Lysozyme at 100 &#181;g/mL can be added to enhance bacteria lysis.</li>
	<li>Incubate the bacteria lysate on ice for 15 min. Use a probe sonicator and sonicate at 70% output amplitude for 30 s (4&#8211;5x), and then centrifuge at 21,000 x g for 20 min at 4 &#176;C. 
	CAUTION: During sonication, keep always the samples on ice.</li>
	<li>During the time of centrifugation, take 500 &#181;L packed GSH-agarose beads (50% slurry) in a 15 mL tube and add 10 mL of buffer A without PMSF and anti-proteases. Mix briefly and spin at 2,500 g for 3 min at 4 &#176;C, repeat this wash step two more times and keep the beads on ice.</li>
	<li>Following bacterial lysate centrifugation, collect the supernatant and transfer it into a 50 mL conical tube. Take an aliquot of 100 &#956;L as a total lysate and add 100 &#956;L of 2x Laemmli sample buffer for SDS-PAGE and Coomassie blue analysis.</li>
	<li>Filter the lysate through 0.45 &#956;m pore syringe filter and mix it with the GSH beads. Incubate at 4 &#176;C with shaking for 5 h. Spin down the beads at 2,500 x g for 3 min, remove, and save the supernatant as the flow through. Wash the proteins-GSH bound beads 6 times (5 mL each) with buffer A containing anti-protease cocktail and PMSF.</li>
	<li>After the last wash, transfer the beads along with 10 mL of buffer into an empty chromatography column, add 1.5 mL buffer A containing 25 mM glutathione, and collect the elution by gravity into 1.5 mL microtubes. Repeat the elution procedure 4 times. 
	NOTE: Glutathione stock solution is prepared at 200 mM concentration in 50 mM Tris-HCl, pH 7.5</li>
	<li>Take an aliquot of 10 &#181;L from each one of the 4 elutions and add 10 &#181;L of 2x Laemmli sample buffer. These samples will be used for SDS-PAGE and Coomassie blue analysis to determine the presence and the purity of the purified proteins. 
	NOTE: Following the last elution, keep the beads in buffer at 4 &#176;C for recycling and future use.</li>
	<li>Choose the eluted fractions showing reasonable amounts of purified proteins for further analysis. Make small aliquots of the preparation. Store the purified proteins in -80&#176;C. Usually, protein concentration will range from 0.5 &#181;g to 2 &#181;g per &#181;L and samples can be stored in this state.</li>
	<li>OPTIONAL: Concentrate the sample or change buffer using a 10K centrifugal filter unit</li>
</ol>
2. Purification of the BAP1/DEUBAD (ASXL2) Deubiquitinase Complex
<ol>
	<li>Use pET30a+-His-BAP138 and pDEST-MBP-DEUBAD (ASXL2)35 bacterial expression constructs to transform BL21 (DE3) RIL bacteria for the production of His-BAP1 and MBP-DEUBAD respectively.</li>
	<li>Perform two separate overnight starter cultures by incubating His-BAP1 and MBP-DEUBAD (ASXL2) expressing bacteria in 20 mL of LB broth of ampicillin medium containing 50 &#181;g/mL chloramphenicol and the corresponding antibiotic for each plasmid, 100 &#181;g/mL of kanamycin or 100 &#181;g/mL of ampicillin respectively. Place on shaker at 37&#176;C.</li>
	<li>Perform steps 1.3&#173;&#8211;1.6.</li>
	<li>Resuspend the bacteria pellets in 25 mL of ice-cold lysis buffer B (50 mM Tris pH 7.3, 500 mM NaCl, 3 mM &#946;-Mercaptoethanol, 0.2% Triton X-100, 1 mM PMSF and 1/100 anti-protease cocktail). Mix equal volumes of the His-BAP1 with the MBP-DEUBAD lysates and incubate on ice for 15 min. Sonicate and then centrifuge the lysate as indicated in protocol 1 (step 9).
	<ol>
		<li>During the centrifugation, prepare 500 &#181;L packed Ni-NTA agarose beads (50% slurry) by washing them three times with buffer B without PMSF and anti-protease cocktail.</li>
	</ol>
	</li>
	<li>Following bacterial lysate centrifugation, collect the supernatant and transfer it into a 50 mL conical tube. Take an aliquot of 100 &#956;L as a total lysate and add 100 &#956;L of 2x Laemmli sample buffer&#160;for SDS-PAGE and Coomassie blue analysis.</li>
	<li>Filter the lysate through 0.45 &#956;m pore syringe filter and mix it with the Ni-NTA beads. Incubate at 4&#176;C with shaking for 5 h. Spin down the beads at 2,500 x g for 3 min, remove, and save the supernatant as the flow through.
	<ol>
		<li>Wash the beads 6 times (5 mL each) with buffer B containing 20 mM 1,3-Diaza-2,4-cyclopentadiene (Imidazole). 
		NOTE: Make a stock solution of 2 M imidazole at pH 7.3.</li>
	</ol>
	</li>
	<li>After the last wash, transfer the beads with 10 mL of buffer into an empty chromatography column, add 1 mL buffer B containing 200 mM Imidazole and collect the elution by gravity into 1.5 mL microtubes containing 10 &#181;L DTT (500 mM) and 2 &#181;L EDTA (500 mM, pH: 8). Repeat the elution procedure 4 times. Take 10 &#181;L of each elution fraction and add 10 &#181;L of 2x Laemmli sample buffer for SDS-PAGE and Coomassie blue analysis. Pool the desired eluted fractions. 
	NOTE: Keep the beads in buffer at 4 &#176;C for recycling and future use.</li>
	<li>Dilute the eluted complex 3 times with buffer C (50 mM Tris pH 7.3, 300 mM NaCl, 1% Triton X-100, 5 mM DTT, 1 mM EDTA, 1 mM PMSF, and 1/100 protease inhibitor cocktail) prior to incubation with the amylose agarose beads.
	<ol>
		<li>Prepare amylose agarose beads (500 &#181;L packed) by washing them 2 times with the buffer C without adding PMSF and anti-protease cocktail. Add the beads to the diluted elution and incubate overnight at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Centrifuge at 2,500 x g for 3 min and keep the flow through. Wash the proteins-bounds beads with 5 mL of buffer C (5&#8211;6x).</li>
	<li>Keep the purified His-BAP1/MBP-DEUBAD complex on the amylose agarose beads in buffer D (50 mM HEPES pH 8.0, 50 mM NaCl, 10% Glycerol, and 1 mM DTT) and store at -80 &#176;C. Take 20 &#181;L of the beads fraction (50% beads solution) and add 20 &#181;L of 2x Laemmli sample buffer for SDS-PAGE and Coomassie blue analysis.</li>
</ol>
3. Purification of the Nucleosomes from HEK293T Cells
<ol>
	<li>Culture HEK293T cells in complete Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 5% newborn calf serum (NBS), 2 mM L-glutamine, and 1% penicillin/streptomycin.</li>
	<li>Plate around 12 x 106 HEK293T cells in 20 mL of complete media per 15 cm culture dish one day before the transfection (10 dishes were used). Before transfection, change the cell&#8217;s medium&#160;to 12 mL of serum-free medium&#160;and transfect the cells with 21 &#181;g of pCDNA-Flag-H2A using 63 &#181;l of polyethylenimine (PEI) at 1 mg/mL36. Change to complete medium 12 h later.</li>
	<li>Three days post-transfection, wash the cells with 15 mL ice-cold PBS (twice) and harvest them in 3 mL of ice-cold PBS using a cell scraper. Centrifuge at 2,100 x g for 8 min at 4 &#176;C. Discard the PBS and proceed to the lysis step or freeze the cell pellet at -80 &#176;C.</li>
	<li>Resuspend the cell pellet in about 10 volumes of buffer E (50 mM Tris-HCl pH 7.3, 420 mM NaCl, 1% NP-40, 1 mM MgCl2, 1 mM PMSF, 1/100 protease inhibitor cocktail, and 20 mM of N-methylmaleimide (NEM)) and incubate on ice for 20 min. Adding N-methylmaleimide (NEM) is critical to inhibit DUBs that co-purify with nucleosomes.</li>
	<li>After centrifugation at 3,000 x g for 5 min, discard the supernatant and resuspend the chromatin pellet in 10 volumes of buffer E. Mix by inversion and centrifuge at 3,000 x g for 5 min.
	<ol>
		<li>Repeat the wash step another time using buffer E and two times using buffer F &#34;MNase Buffer&#34; (20 mM Tris-HCl pH 7.5, 100 mM KCl, 2 mM MgCl2, 1 mM CaCl2, 0.3 M sucrose, 0.1% NP-40, 1 mM PMSF, and 1/100 protease inhibitor cocktail). 
		CAUTION: Pellet will float during washes and centrifugation.</li>
	</ol>
	</li>
	<li>Resuspend the chromatin in 5 mL buffer F and treat the pellet with Micrococcal nuclease (MNase) (3 U/mL for 10 min at room temperature). 
	NOTE: The reaction can be aided by mixing using a Dounce Homogenizer.</li>
	<li>After incubation, take a 40 &#956;L aliquot of the mixture for analysis of nucleosomal DNA fragments. Mix this aliquot with 40 &#956;L of Phenol/chloroform and 20 &#956;L of 6x DNA loading buffer, vortex, spin at 18,000 x g for 2 min, and load the DNA on a 2% agarose gel.
	<ol>
		<li>When the DNA is predominantly a 147 bp fragment corresponding to mononucleosomes, stop the reaction by adding 5 mM EDTA at final concentration.</li>
	</ol>
	</li>
	<li>Following centrifugation at 21,000 x g for 20 min at 4&#176;C, incubate the soluble chromatin fraction with anti-Flag resin overnight at 4 &#176;C. Wash the beads with 6x Buffer G (50 mM Tris-HCl, pH 7.3, 5 mM EDTA, 150 mM NaCl, 1% NP-40, 1 mM PMSF, 1 mM DTT, 1/100 protease inhibitors cocktail).</li>
	<li>Transfer the beads with 5 mL buffer G into an empty chromatography column. Elute the beads-bound nucleosomes with 200 &#956;g/mL of Flag elution peptide. Use an elution buffer composed of buffer G containing 200 &#956;g/mL Flag elution peptides (see Table of Materials) and 1/5 (vol:vol) 1 M Tris, pH 8.0. Elute the nucleosomes 3x with 260 &#956;L Flag elution Buffer (2 h each elution).</li>
	<li>Test the 3 elutions by taking an aliquot for phenol-chloroform extraction and load the DNA in a 2% agarose gel. Take 10 &#181;L of each elution fraction and add 10 &#181;L of Laemmli sample buffer 2x for SDS-PAGE and Coomassie blue analysis and load each elution for Western Blot detection of ubiquitinated H2A. Store the elution at -80 &#176;C. In general, purification from human cells yields less amount of proteins than that from bacteria, with concentrations ranging from 0.1 to 0.5 &#956;g/&#956;L.</li>
</ol>
4. Nucleosome Ubiquitination Assay Using BMI1/RING1B E3 Ubiquitin Ligase Complex
<ol>
	<li>Centrifuge the nucleosomes through 10K pore 0.5 mL centrifugal filters to change the substrate from the elution buffer to the reaction buffer H (25 mM Tris, pH 7.5; 10 mM MgCl2, and 5 mM ATP). Alternatively, commercially available nucleosomes can be used for the assay. 
	NOTE: Changing substrate suspension solution to reaction buffer ensures reproducibility and prevents potential E3 ligase inhibition by compounds present in the Flag elution mixture.</li>
	<li>Incubate 1 &#956;g of the nucleosomes in 40 &#956;L total volume of buffer H. Add Ub Activating Enzyme (UBE1) (250 ng), Ub (50 ng/&#956;L) and E2 Ub-conjugating enzymes (672 ng), and 1 &#956;g of BMI1/RING1B E3 ubiquitin ligase complex. Incubate the reaction for 3 h at 37 &#176;C with occasional shaking. 
	NOTE: Several controls can be conducted in parallel including omitting, E1, E2, E3, ATP and ubiquitin. This ensures the specificity for the ubiquitination reaction.</li>
	<li>Stop the reaction by adding 40 &#956;L of 2x Laemmli sample buffer and analyze histone H2A K119 ubiquitination by SDS-PAGE and western blotting, according to standard procedures. Use either anti-H2A or anti-H2A K119ub antibodies (see Table of Materials).</li>
</ol>
5. In Vitro Nucleosomal H2A DUB Assay Using BAP1/DEUBAD
<ol>
	<li>Use the purified nucleosomes for&#160;the in vitro deubiquitination assay. Resuspend 100&#8211;500 ng of nucleosomes in 40 &#956;L buffer I (50 mM Tris-HCl, pH 7.3, 1 mM MgCl2, 50 mM NaCl, and 1 mM DTT). Add 1 &#956;g of BAP1 bacteria-purified His-BAP1 and MBP-DEUBAD. Carry out the deubiquitination reaction for 3 h at 37 &#176;C with occasional shaking.</li>
	<li>Stop the in vitro reaction by adding 40 &#956;L of 2x Laemmli buffer and analyze by immunoblotting.</li>
</ol>

<ol>
	<li>Ye, Y., Rape, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+ubiquitin+chains:+E2+enzymes+at+work.">Building ubiquitin chains: E2 enzymes at work.</a> Nature Reviews Molecular Cell Biology. 10, 755-764 (2009).</li><li>Komander, D., Clague, M. J., Urbe, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+the+chains:+structure+and+function+of+the+deubiquitinases.">Breaking the chains: structure and function of the deubiquitinases.</a> Nature Reviews Molecular Cell Biology. 10, 550-563 (2009).</li><li>Ciechanover, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+unravelling+of+the+ubiquitin+system.">The unravelling of the ubiquitin system.</a> Nature Reviews Molecular Cell Biology. 16, 322-324 (2015).</li><li>Nakayama, K. I., Nakayama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin+ligases:+cell-cycle+control+and+cancer.">Ubiquitin ligases: cell-cycle control and cancer.</a> Nature Reviews Cancer. 6, 369-381 (2006).</li><li>Senft, D., Qi, J., Ronai, Z. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin+ligases+in+oncogenic+transformation+and+cancer+therapy.">Ubiquitin ligases in oncogenic transformation and cancer therapy.</a> Nature Reviews Cancer. 18, 69-88 (2018).</li><li>Zheng, N., Shabek, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin+Ligases:+Structure,+Function,+and+Regulation.">Ubiquitin Ligases: Structure, Function, and Regulation.</a> Annual Review of Biochemistry. 86, 129-157 (2017).</li><li>Iwai, K., Fujita, H., Sasaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linear+ubiquitin+chains.+NF-kappaB+signalling,+cell+death+and+beyond.">Linear ubiquitin chains. NF-kappaB signalling, cell death and beyond.</a> Nature Reviews Molecular Cell Biology. 15, 503-508 (2014).</li><li>Kulathu, Y., Komander, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atypical+ubiquitylation+-+the+unexplored+world+of+polyubiquitin+beyond+Lys48+and+Lys63+linkages.">Atypical ubiquitylation - the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages.</a> Nature Reviews Molecular Cell Biology. 13, 508-523 (2012).</li><li>Yau, R., Rape, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+increasing+complexity+of+the+ubiquitin+code.">The increasing complexity of the ubiquitin code.</a> Nature Cell Biology. 18, 579-586 (2016).</li><li>Mevissen, T. E. T., Komander, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+Deubiquitinase+Specificity+and+Regulation.">Mechanisms of Deubiquitinase Specificity and Regulation.</a> Annual Review of Biochemistry. 86, 159-192 (2017).</li><li>Bedford, L., Lowe, J., Dick, L. R., Mayer, R. J., Brownell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin-like+protein+conjugation+and+the+ubiquitin-proteasome+system+as+drug+targets.">Ubiquitin-like protein conjugation and the ubiquitin-proteasome system as drug targets.</a> Nature Reviews Drug Discovery. 10, 29-46 (2011).</li><li>Minton, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammasomes:+Ubiquitin+lines+up+for+inflammasome+activity.">Inflammasomes: Ubiquitin lines up for inflammasome activity.</a> Nature Reviews Immunology. 14, 580-581 (2014).</li><li>Popovic, D., Vucic, D., Dikic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitination+in+disease+pathogenesis+and+treatment.">Ubiquitination in disease pathogenesis and treatment.</a> Nature Medicine. 20, 1242-1253 (2014).</li><li>Upadhyay, A., Amanullah, A., Chhangani, D., Mishra, R., Mishra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+multifaceted+E3+ubiquitin+ligases+barricade+extreme+defense:+Potential+therapeutic+targets+for+neurodegeneration+and+ageing.">Selective multifaceted E3 ubiquitin ligases barricade extreme defense: Potential therapeutic targets for neurodegeneration and ageing.</a> Ageing Research Reviews. 24, 138-159 (2015).</li><li>Hammond-Martel, I., Yu, H., Affar el, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+ubiquitin+signaling+in+transcription+regulation.">Roles of ubiquitin signaling in transcription regulation.</a> Cellular Signalling. 24, 410-421 (2012).</li><li>Schwertman, P., Bekker-Jensen, S., Mailand, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+DNA+double-strand+break+repair+by+ubiquitin+and+ubiquitin-like+modifiers.">Regulation of DNA double-strand break repair by ubiquitin and ubiquitin-like modifiers.</a> Nature Reviews Molecular Cell Biology. 17, 379-394 (2016).</li><li>Uckelmann, M., Sixma, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+ubiquitination+in+the+DNA+damage+response.">Histone ubiquitination in the DNA damage response.</a> DNA Repair. 56, 92-101 (2017).</li><li>Robzyk, K., Recht, J., Osley, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rad6-dependent+ubiquitination+of+histone+H2B+in+yeast.">Rad6-dependent ubiquitination of histone H2B in yeast.</a> Science. 287, 501-504 (2000).</li><li>Hwang, W. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+RING+finger+protein+required+for+histone+H2B+monoubiquitination+and+cell+size+control.">A conserved RING finger protein required for histone H2B monoubiquitination and cell size control.</a> Molecular Cell. 11, 261-266 (2003).</li><li>Kim, J., Hake, S. B., Roeder, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+homolog+of+yeast+BRE1+functions+as+a+transcriptional+coactivator+through+direct+activator+interactions.">The human homolog of yeast BRE1 functions as a transcriptional coactivator through direct activator interactions.</a> Molecular Cell. 20, 759-770 (2005).</li><li>Wood, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=an+E3+ubiquitin+ligase+required+for+recruitment+and+substrate+selection+of+Rad6+at+a+promoter.">an E3 ubiquitin ligase required for recruitment and substrate selection of Rad6 at a promoter.</a> Molecular Cell. 11, 267-274 (2003).</li><li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+histone+H2A+ubiquitination+in+Polycomb+silencing.">Role of histone H2A ubiquitination in Polycomb silencing.</a> Nature. 431, 873-878 (2004).</li><li>Buchwald, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+E3-ligase+activity+of+the+Ring-Ring+complex+of+polycomb+proteins+Bmi1+and+Ring1B.">Structure and E3-ligase activity of the Ring-Ring complex of polycomb proteins Bmi1 and Ring1B.</a> The EMBO Journal. 25, 2465-2474 (2006).</li><li>Scheuermann, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+H2A+deubiquitinase+activity+of+the+Polycomb+repressive+complex+PR-DUB.">Histone H2A deubiquitinase activity of the Polycomb repressive complex PR-DUB.</a> Nature. 465, 243-247 (2010).</li><li>Jensen, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAP1:+a+novel+ubiquitin+hydrolase+which+binds+to+the+BRCA1+RING+finger+and+enhances+BRCA1-mediated+cell+growth+suppression.">BAP1: a novel ubiquitin hydrolase which binds to the BRCA1 RING finger and enhances BRCA1-mediated cell growth suppression.</a> Oncogene. 16, 1097-1112 (1998).</li><li>Harbour, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequent+mutation+of+BAP1+in+metastasizing+uveal+melanomas.">Frequent mutation of BAP1 in metastasizing uveal melanomas.</a> Science. 330, 1410-1413 (2010).</li><li>Abdel-Rahman, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GermLine+BAP1+mutation+predisposes+to+uveal+melanoma,+lung+adenocarcinoma,+meningioma,+and+other+cancers.">GermLine BAP1 mutation predisposes to uveal melanoma, lung adenocarcinoma, meningioma, and other cancers.</a> Journal of Medical Genetics. , (2011).</li><li>Bott, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nuclear+deubiquitinase+BAP1+is+commonly+inactivated+by+somatic+mutations+and+3p21.1+losses+in+malignant+pleural+mesothelioma.">The nuclear deubiquitinase BAP1 is commonly inactivated by somatic mutations and 3p21.1 losses in malignant pleural mesothelioma.</a> Nature Genetics. 43, 668-672 (2011).</li><li>Goldstein, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GermLine+BAP1+mutations+and+tumor+susceptibility.">GermLine BAP1 mutations and tumor susceptibility.</a> Nature Genetics. 43, 925-926 (2011).</li><li>Testa, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GermLine+BAP1+mutations+predispose+to+malignant+mesothelioma.">GermLine BAP1 mutations predispose to malignant mesothelioma.</a> Nature Genetics. 43, 1022-1025 (2011).</li><li>Wiesner, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GermLine+mutations+in+BAP1+predispose+to+melanocytic+tumors.">GermLine mutations in BAP1 predispose to melanocytic tumors.</a> Nature Genetics. 43, 1018-1021 (2011).</li><li>Dey, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+the+tumor+suppressor+BAP1+causes+myeloid+transformation.">Loss of the tumor suppressor BAP1 causes myeloid transformation.</a> Science. 337, 1541-1546 (2012).</li><li>Pena-Llopis, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAP1+loss+defines+a+new+class+of+renal+cell+carcinoma.">BAP1 loss defines a new class of renal cell carcinoma.</a> Nature Genetics. 44, 751-759 (2012).</li><li>Bononi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAP1+regulates+IP3R3-mediated+Ca2++flux+to+mitochondria+suppressing+cell+transformation.">BAP1 regulates IP3R3-mediated Ca2+ flux to mitochondria suppressing cell transformation.</a> Nature. 546, 549-553 (2017).</li><li>Daou, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoubiquitination+of+ASXLs+controls+the+deubiquitinase+activity+of+the+tumor+suppressor+BAP1.">Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1.</a> Nature Communications. 9, 4385 (2018).</li><li>Daou, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BAP1/ASXL2+Histone+H2A+Deubiquitinase+Complex+Regulates+Cell+Proliferation+and+Is+Disrupted+in+Cancer.">The BAP1/ASXL2 Histone H2A Deubiquitinase Complex Regulates Cell Proliferation and Is Disrupted in Cancer.</a> The Journal of Biological Chemistry. 290, 28643-28663 (2015).</li><li>Mashtalir, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autodeubiquitination+protects+the+tumor+suppressor+BAP1+from+cytoplasmic+sequestration+mediated+by+the+atypical+ubiquitin+ligase+UBE2O.">Autodeubiquitination protects the tumor suppressor BAP1 from cytoplasmic sequestration mediated by the atypical ubiquitin ligase UBE2O.</a> Molecular Cell. 54, 392-406 (2014).</li><li>Yu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin+carboxyl+hydrolase+BAP1+forms+a+ternary+complex+with+YY1+and+HCF-1+and+is+a+critical+regulator+of+gene+expression.">The ubiquitin carboxyl hydrolase BAP1 forms a ternary complex with YY1 and HCF-1 and is a critical regulator of gene expression.</a> Molecular and Cellular Biology. 30, 5071-5085 (2010).</li><li>Yu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+suppressor+and+deubiquitinase+BAP1+promotes+DNA+double-strand+break+repair.">Tumor suppressor and deubiquitinase BAP1 promotes DNA double-strand break repair.</a> Proceedings of the National Academy of Sciences of the United States of America. 111, 285-290 (2014).</li><li>Dai, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAP1+inhibits+the+ER+stress+gene+regulatory+network+and+modulates+metabolic+stress+response.">BAP1 inhibits the ER stress gene regulatory network and modulates metabolic stress response.</a> Proceedings of the National Academy of Sciences of the United States of America. 114, 3192-3197 (2017).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAP1+links+metabolic+regulation+of+ferroptosis+to+tumour+suppression.">BAP1 links metabolic regulation of ferroptosis to tumour suppression.</a> Nature Cell Biology. 20, 1181-1192 (2018).</li><li>Kolluri, K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+functional+BAP1+augments+sensitivity+to+TRAIL+in+cancer+cells.">Loss of functional BAP1 augments sensitivity to TRAIL in cancer cells.</a> eLife. 7, (2018).</li><li>Machida, Y. J., Machida, Y., Vashisht, A. A., Wohlschlegel, J. A., Dutta, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+deubiquitinating+enzyme+BAP1+regulates+cell+growth+via+interaction+with+HCF-1.">The deubiquitinating enzyme BAP1 regulates cell growth via interaction with HCF-1.</a> The Journal of Biological Chemistry. , (2009).</li><li>Sahtoe, D. D., van Dijk, W. J., Ekkebus, R., Ovaa, H., Sixma, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAP1/ASXL1+recruitment+and+activation+for+H2A+deubiquitination.">BAP1/ASXL1 recruitment and activation for H2A deubiquitination.</a> Nature Communications. 7, 10292 (2016).</li></ol>

The authors declare no competing financial interests.

There are several advantages of establishing robust in vitro ubiquitination and deubiquitination assays for proteins of interest. These assays can be used to: (i) establish optimal conditions and define minimal requirement for these reactions, (ii) determine enzymatic kinetic and biochemical constants, (iii) define the roles of cofactors or inhibitors that can impact these reactions, (iv) identify interaction interfaces, (v) test the impact of artificial or disease-associated mutations and (vi) establish assay conditions...

Ubiquitination is one of the most conserved post-translational modifications and is critical for a wide variety of organisms including yeast, plants and vertebrates. Ubiquitination consists of the covalent attachment of ubiquitin, a highly conserved 76 amino acid polypeptide, to target proteins and occurs in three sequential steps involving three enzymes, i.e., E1-activating, E2-conjugating and E3 ligase1,2,3. This post-translational modification plays central roles in a wide spectrum of biological processes. Indeed, the E3 ligases, which provide the specificity of the reaction, constitute a large superfamily of enzymes and are the most abundant enzymes of the ubiquitin system4,5,6. The downstream effects of protein ubiquitination depend on nature of the modification: monoubiquitination, multi-monoubiquitination, and linear or branched polyubiquitination. Monoubiquitination is rarely associated with proteasomal degradation, but instead this modification is involved in mediating various signaling events. Polyubiquitination involves the N-terminal or the lysine residues in ubiquitin molecule itself, and the destiny of a polyubiquitinated protein depends on which residue is involved in ubiquitin chain extension. It has long been known that polyubiquitination mediated by lysine 48 of ubiquitin induces proteasomal degradation. On the contrary, polyubiquitination via lysine 63 of ubiquitin is often associated with protein activation7,8,9. Similar to other important post-translational modifications, ubiquitination is reversible and ubiquitin removal from proteins is ensured by specific proteases termed deubiquitinases (DUBs), which have emerged as important regulators of cellular processes2,10. Importantly, many DUBs are highly specialized, and regulate, through deubiquitination, specific substrates, indicating that a fine balance between ubiquitination and deubiquitination is critical for protein function. E3s and DUBs, along with the proteasome degradation machinery and accessory factors, form the Ubiquitin Proteasome System (UPS, with &#62;1200 genes) which regulates major signaling pathways, several of which are associated with cell growth and proliferation, cell fate determination, differentiation, cell migration, and cell death. Importantly, deregulation of several signaling cascades involving ubiquitination promotes tumorigenesis and neurodegeneration diseases5,11,12,13,14.
Ubiquitination plays pervasive roles in chromatin biology and DNA-dependent processes15,16,17. For instance, monoubiquitination of histone H2A on lysine 119 (hereafter H2A K119ub) is a critical post-translational modification involved in transcriptional repression and DNA repair18,19,20,21,22. H2A K119ub is catalyzed by the Polycomb Repressive Complex 1 (PRC1), which plays a key role in the maintenance of epigenetic information and is highly conserved from Drosophila to human. Canonical PRC1 is constituted notably by the RING1B and BMI1, which are the core E3 ubiquitin ligase complex responsible for the above-mentioned ubiquitination event22,23. In Drosophila, H2A monoubiquitination (H2A K118ub which corresponds to H2A K119ub in mammalians) is reversed by the DUB Calypso, which interacts with Additional Sex Comb (ASX) forming the Polycomb-repressive DUB (PR-DUB) complex24. The mammalian ortholog of calypso, BAP1, is a tumor suppressor deleted or inactivated in various human malignancies25,26,27,28,29,30,31,32,33. BAP1 regulates DNA-dependent processes in the nucleus and Calcium-signaling-mediated apoptosis at the endoplasmic reticulum33,34,35,36,37,38,39,40,41,42. BAP1 assembles multi-subunit protein complexes containing transcription regulators notably ASXL1, ASXL2 and ASXL3 (ASXLs), three orthologues of ASX38,43. ASXLs use the DEUBiquitinase ADaptor (DEUBAD) domain, also termed ASXM domain, to stimulate BAP1 DUB activity35,36,44. Hence, ASXLs play important roles in coordinating BAP1 DUB activity at chromatin and more broadly its tumor suppressor function.
Several methods exist to study ubiquitination and deubiquitination processes. Notably, biochemical assays using proteins purified from bacteria remain very powerful in demonstrating direct ubiquitination of, or removal of ubiquitin from, specific substrates. These experiments can be conducted to investigate a range of parameters such as the determining the requirement of minimal complexes, determining reactions kinetics, defining structure/function relationships, and understanding the impact of pathological gene mutations. Here, we provide protocols to conduct ubiquitination and deubiquitination reactions on chromatin substrates with purified components. As a model system, in vitro ubiquitination and deubiquitination of nucleosomal H2A protein is presented. Bacteria-purified proteins assembled in minimal complexes of RING1B/BMI1 and BAP1/DEUBAD are used for ubiquitination or deubiquitination of nucleosomal H2A, respectively.

<table>
	<tbody>
		<tr>
			<td>Amylose agarose beads</td>
			<td>New England Biolabs</td>
			<td>#E8021</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra 0.5 ml centrifugal filters 10K</td>
			<td>Sigma-Aldrich</td>
			<td>#UFC501096</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-H2AK119ub (H2Aub)</td>
			<td>Cell Signaling Technology</td>
			<td>#8240</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Flag-agarose beads</td>
			<td>Sigma-Aldrich</td>
			<td>#A4596</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-protease cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>#P8340</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 (DE3) CodonPlus-RIL bacteria</td>
			<td>Agilent technologies</td>
			<td>#230240</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Wisent</td>
			<td>#319-005-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Empty chromatography column</td>
			<td>Biorad</td>
			<td>#731-1550</td>
			<td></td>
		</tr>
		<tr>
			<td>Flag peptide</td>
			<td>Sigma-Aldrich</td>
			<td>#F3290</td>
			<td></td>
		</tr>
		<tr>
			<td>GSH-agarose beads</td>
			<td>Sigma-Aldrich</td>
			<td>#G4510</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T</td>
			<td>ATCC</td>
			<td>#CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>#I5513</td>
			<td></td>
		</tr>
		<tr>
			<td>Micrococcal nuclease (MNase)</td>
			<td>Sigma-Aldrich</td>
			<td>#N3755</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA agarose beads</td>
			<td>ThermoFisher Scientific</td>
			<td>#88221</td>
			<td></td>
		</tr>
		<tr>
			<td>N-methylmaleimide (NEM)</td>
			<td>Bioshop</td>
			<td>#ETM222</td>
			<td></td>
		</tr>
		<tr>
			<td>Pore syringe filter 0.45 &#956;m</td>
			<td>Sarstedt</td>
			<td>#83.1826</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine (PEI)</td>
			<td>Polysciences Inc</td>
			<td>#23966-1</td>
			<td></td>
		</tr>
		<tr>
			<td>pGEX6p2rbs-GST-RING1B(1-159)-Bmi1(1-109)</td>
			<td>Addgene</td>
			<td>#63139</td>
			<td></td>
		</tr>
		<tr>
			<td>Ub Activating Enzyme (UBE1)</td>
			<td>Boston Biochem</td>
			<td>#E-305</td>
			<td></td>
		</tr>
		<tr>
			<td>UBCH5C (UBE2D3)</td>
			<td>Boston Biochem</td>
			<td>#E2-627</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro ubiquitination and deubiquitination assays of nucleosomal histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

GST-BMI1 and RING1B proteins are well produced in bacteria and can be readily extracted in the soluble fraction. Figure 1A shows a Coomassie blue staining for a typical purification of the GST-BMI1-RING1B complex. The GST-BMI1 and RING1B bands migrate at the expected molecular weight, ~45 kDa and ~13 kDa respectively. Notably the E3 ligase complex is highly homogenous with very low levels of bacteria proteins contaminants and/or degradation products. Moreover...

Watch this Scientific Journal Video about In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones at JoVE.com

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

in-vitro-ubiquitination-deubiquitination-assays-nucleosomal

Research

JoVE Journal

Biochemistry

10.5K Views.  University of Montréal. Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

Video: In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications (PTMs). They are newly identified species with unknown means of establishment and functional implications. Current analytical methods are inadequate to detect the copy-specific occurrence of PTMs on the nucleosomal sister histones. This protocol presents a biochemical method for the in vitro reconstitution of nucleosomes containing differentially isotope-labeled sister histones. The generated complex can be also asymmetrically modified, after including a premodified histone pool during refolding of histone subcomplexes. These asymmetric nucleosome preparations can be readily reacted with histone-modifying enzymes to study modification cross-talk mechanisms imposed by the asymmetrically pre-incorporated PTM using nuclear magnetic resonance (NMR) spectroscopy. Particularly, the modification reactions in real-time can be mapped independently on the two sister histones by performing different types of NMR correlation experiments, tailored for the respective isotope type. This methodology provides the means to study crosstalk mechanisms that contribute to the formation and propagation of asymmetric PTM patterns on nucleosomal complexes.

This protocol describes the reconstitution of nucleosomes containing differentially isotope-labeled sister histones. At the same time, asymmetrically post-translationally modified nucleosomes can be generated after using a premodified histone copy. These preparations can be readily used to study modification crosstalk mechanisms, simultaneously on both sister histones, by using high-resolution NMR spectroscopy.

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications ...

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.

This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine whether there is an imbalance in age-related energy metabolism between mitochondrial oxidative phosphorylation and aerobic glycolysis. Here, we show the utilization of commercial colorimetric assay kits for lactate and pyruvate in the model organism C. elegans. Recently, the sensitivity and accuracy of the colorimetric/fluorimetric assay kits have been improved greatly by the research and development conducted by reagent manufacturers. The improved reagents have enabled the use of small-scale assays with a 96-well plate in C. elegans. In general, a fluorimetric assay is superior in sensitivity to a colorimetric assay; however, the colorimetric approach is more suitable for the use in common laboratories. Another important issue in these assays for quantitative determination is protein precipitation of homogenized C. elegans samples. In our protein precipitation method, common precipitants (e.g., trichloroacetic acid, perchloric acid and metaphosphoric acid) are used for sample preparation. A protein-free assay sample is prepared by directly adding cold precipitant (final concentration of 5%) during homogenization.

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

We describe a modified small-scale extraction and colorimetric assays of lactate and pyruvate in the nematode C. elegans. When utilizing commercial assay kits, the technical development of their sensitivity and accuracy is important. Protein precipitation in extraction is the most critical step for the quantitative determination of intracellular metabolites.

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within the chloroplasts, the thylakoid membrane system houses all the photosynthetic pigments, reaction center complexes, and most of the electron carriers, and is responsible for light-dependent ATP synthesis. Over 90% of chloroplast proteins are encoded in the nucleus, translated in the cytosol, and subsequently imported into the chloroplast. Further protein transport into or across the thylakoid membrane utilizes one of four translocation pathways. Here, we describe a high-yield method for isolation of transport-competent thylakoids from peas (Pisum sativum), along with transport assays through the three energy-dependent cpTat, cpSec1, and cpSRP-mediated pathways. These methods enable experiments relating to thylakoid protein localization, transport energetics, and the mechanisms of protein translocation across biological membranes.

We present protocols herein for high-yield isolation of physiologically active thylakoids and protein transport assays for the chloroplast twin arginine translocation (cpTat), secretory (cpSec1), and signal recognition particle (cpSRP) pathways.

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach provides real-time cell monitoring with a device-dependent time resolution down to several tens of milliseconds and it is highly automated. However, when sample numbers get high (e.g., dose-response studies for various different ligands), the cost for the disposable electrode arrays as well as the available time resolution for sequential well-by-well recordings may become limiting. Therefore, we here present a serial agonist addition protocol which has the potential to significantly increase the output of label-free GPCR assays. Using the serial agonist addition protocol, a GPCR agonist is added sequentially in increasing concentrations to a single cell layer while continuously monitoring the sample&#39;s impedance (agonist mode). With this serial approach, it is now possible to establish a full dose-response curve for a GPCR agonist from just one single cell layer. The serial agonist addition protocol is applicable to different GPCR coupling types, Gq Gi/0 or Gs and it is compatible with recombinant and endogenous expression levels of the receptor under study. Receptor blocking by GPCR antagonists is assessable as well (antagonist mode).

This protocol demonstrates real-time recording of full dose-response relationships for agonist-induced GPCR activation from a single cell layer grown on a single microelectrode using label-free impedance measurements. The new dosing scheme significantly increases throughput without loss in time resolution.

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach ...

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi pyrrolysine tRNA-synthetase (MmAcKRS1) and its cognate tRNA (tRNAPyl) to assemble reconstituted mononucleosomes with site specific acetylated histones. MmAcKRS1 and tRNAPyl deliver AcK at an amber mutation site in the mRNA of choice during translation in Escherichia coli. This technique has been used extensively to incorporate AcK at H3 lysine sites. Pyrrolysyl-tRNA synthetase (PylRS) can also be easily evolved to incorporate other noncanonical amino acids (ncAAs) for site specific protein modification or functionalization. Here we detail a method to incorporate AcK using the MmAcKRS1 system into histone H3 and integrate acetylated H3 proteins into reconstituted mononucleosomes. Acetylated reconstituted mononucleosomes can be used in biochemical and binding assays, structure determination, and more. Obtaining modified mononucleosomes is crucial for designing experiments related to discovering new interactions and understanding epigenetics.

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Here we present a method to express acetylated histone proteins using genetic code expansion and assemble reconstituted nucleosomes in vitro.

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi ...

Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic properties are adapted for the particular function they execute in cells. Myosin 5a processively walks on F-actin to transport melanosomes and vesicles in cells. Conversely, nonmuscle myosin 2b operates as a bipolar filament containing approximately 30 molecules. It moves F-actin of opposite polarity toward the center of the filament, where the myosin molecules work asynchronously to bind actin, impart a power stroke, and dissociate before repeating the cycle. Nonmuscle myosin 2b, along with its other nonmuscle myosin 2 isoforms, has roles that include&#160;cell adhesion, cytokinesis, and tension maintenance. The mechanochemistry of myosins can be studied by performing in vitro motility assays using purified proteins. In the gliding actin filament assay, the myosins are bound to a microscope coverslip surface and translocate fluorescently labeled F-actin, which can be tracked. In the single molecule/ensemble motility assay, however, F-actin is bound to a coverslip and the movement of fluorescently labeled myosin molecules on the F-actin is observed. In this report, the purification of recombinant myosin 5a from Sf9 cells using affinity chromatography is outlined. Following this, we outline two fluorescence microscopy-based assays: the gliding actin filament assay and the inverted motility assay. From these assays, parameters such as actin translocation velocities and single molecule run lengths and velocities can be extracted using the image analysis software. These techniques can also be applied to study the movement of single filaments of the nonmuscle myosin 2 isoforms, discussed herein in the context of nonmuscle myosin 2b. This workflow represents a protocol and a set of quantitative tools that can be used to study the single molecule and ensemble dynamics of nonmuscle myosins.

Presented here is a procedure to express and purify myosin 5a followed by a discussion of its characterization, using both ensemble and single molecule in vitro fluorescence microscopy-based assays, and how these methods can be modified for the characterization of nonmuscle myosin 2b.

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic ...