We would like to thank the Lee lab of the University of Minnesota for providing the mouse spinal cord tissue samples that were used. This work was supported by the Department of Defense Ovarian Cancer Research Program (OCRP) OC093424 to MB, by the Randy Shaver Cancer Research and Community Fund to MB and by the Minnesota Ovarian Cancer Alliance (MOCA) to MB. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

1. Lysis Buffer Preparation
<ol>
	<li>Dissolve sucrose in deionized (DI) water to make a 2 M stock solution. Filter a 2 M solution of sucrose using a vacuum driven 0.22 &#181;m polyvinylidene fluoride (PVDF) filter.</li>
	<li>Dissolve dithiothreitol (DTT) in DI water to make a 500 mM stock solution and store under anaerobic conditions. Dissolve magnesium chloride (MgCl2) in DI water to make a 100 mM stock solution. Dissolve adenosine triphosphate (ATP) disodium hydrate in DI water to make a 50 mM stock solution.</li>
	<li>Make up a Tris solution at pH 7.4 by dissolving Trizma hydrochloride in DI water and adjusting the pH using sodium hydroxide (NaOH).</li>
	<li>Combine portions of the stock solutions in DI water to make a lysis buffer with a sucrose concentration of 250 mM, a DTT concentration of 1 mM, a MgCl2 concentration of 5 mM, an ATP concentration of 1 mM and a Tris concentration of 50 mM. For example, mix 535.3 &#181;l of tris, 500 &#181;l of MgCl2, 1.25 ml of sucrose, 20 &#181;l of DTT, 200 &#181;l of ATP and 7.4947 ml of DI water to make 10 ml of lysis buffer. This can be used for approximately 20 experiments.</li>
</ol>
2. Culturing Cells for the Experiment
<ol>
	<li>Make media by aliquoting 30 ml of DMEM + 10% fetal bovine serum (FBS) into a 50 ml conical tube.</li>
	<li>Transfer frozen M17 or HeLa cells from liquid nitrogen storage to a small beaker with lukewarm water.</li>
	<li>Transfer the cells to the 50 ml conical tube. First, add some media to the cell vial. Then transfer the resuspended cells within 5 sec of thawing.</li>
	<li>Centrifuge the cell suspension at 450 x g for 5 min to obtain a cell pellet. Add 20 ml of media into a T75 flask.</li>
	<li>Remove the media from the cells using suction. Add 5 ml of fresh media to the cell pellet and resuspend.</li>
	<li>Transfer the 5 ml cell suspension to the 20 ml of media in the T75 flask and incubate at 37 &#176;C. Change the media every 3 days.</li>
</ol>
3. Cell Harvesting
<ol>
	<li>Remove the media via suction, being careful not to touch the cells.</li>
	<li>Wash the cells with 5 ml of phosphate buffered saline (PBS). Remove PBS using suction.</li>
	<li>Add 3 ml of trypsin-ethylenediaminetetraacetic acid (EDTA) to the T75 flask and incubate at 37 &#176;C for 3 min.</li>
	<li>Add 6 ml of fresh media to the flask and resuspend the cells.</li>
	<li>Transfer the suspension to a 15 ml conical tube and centrifuge at 290 &#215; g for 5 mins.</li>
	<li>Remove the supernatant and add 5 ml of PBS. Gently tap the bottom of the tube to break up the pellet. Centrifuge at 290 x g for 5 min.</li>
	<li>Repeat step 3.6 three times.</li>
	<li>Remove PBS and resuspend the cells in 1 ml of fresh PBS. Transfer to the Eppendorf tube (1.5 ml). Spin at 290 x g for 5 min.</li>
</ol>
4. Cell Lysis
<ol>
	<li>Measure the approximate volume of the pellet using a pipette and weigh out glass beads in a mass:volume ratio of 1:1. Add twice the volume of the pellet in lysis buffer. 
	Note: Add the buffer before adding the glass beads.</li>
	<li>Lyse the cells in the Eppendorf tube at the maximum agitation for 30 min at 4 &#176;C by vortexing in a cold room.</li>
	<li>Centrifuge briefly at 200 x g for 30 sec to settle the beads. Collect the supernatant.</li>
	<li>Add the same volume of glass beads as before to the supernatant.</li>
	<li>Vortex once again at maximum agitation for 30 min at 4 &#176;C.</li>
	<li>Centrifuge briefly at 200 x g for 30 sec to settle the glass beads. Collect the supernatant.</li>
	<li>Centrifuge at 5,030 x g for 5 min to remove the nuclei, membranes and unbroken cells. Collect the supernatant, which is the cell lysate.</li>
</ol>
5. Tissue Homogenization
<ol>
	<li>Make a mass:volume of 1:9 solution of mouse spinal cord tissue to lysis buffer. 
	Note: This method is applicable to a variety of different tissue samples.</li>
	<li>Homogenize the tissue using a setting of level 2 on the homogenizer for 30 sec to ensure that there are no chunks remaining.</li>
	<li>Spin down at 5,030 x g for 3 min to remove the nuclei, membranes and unbroken cells.</li>
	<li>Collect the supernatant, which is the lysate.</li>
</ol>
6. Sample Derivatization
<ol>
	<li>Perform a bicinchoninic acid (BCA) in a 96-well plate analysis to determine the protein concentration of the tissue lysate using a colorimetric 96-well plate reader as specified by the Pierce BCA Protein Assay kit protocol.</li>
	<li>Bring an aliquot corresponding to 20 &#181;g of total protein to 50 &#181;l using de-ionized (DI) water.</li>
	<li>Add 2 &#181;l of 1.35 &#181;M of HA-Ub-Vinyl sulfone (VS) to the solution and incubate for 1 hr at 37 &#176;C. This results 20 &#181;g of lysate to 50 nM of probe in the final reaction mixture. This is in large molar excess to ensure tagging of functional DUBs.</li>
	<li>Incubate the sample in Laemmli&#8217;s sample buffer for 5 mins at 95 &#176;C. For a 52 &#181;l reaction volume use 26 &#181;l of 2x sample buffer, 13 &#181;l of 4x sample buffer or 8.87 &#181;l of 6x sample buffer.</li>
	<li>Cool on ice before loading the gel.</li>
</ol>
7. Western Blot
<ol>
	<li>Load a 12-well 4-20% tris-glycine gel with 40 &#181;l of the prepared sample</li>
	<li>Run the gel at 95 V until the ion front reaches the bottom.</li>
	<li>Carry out an overnight transfer of the gel onto a polyvinylidene difluoride (PVDF) membrane.</li>
	<li>Incubate the membrane in the amido black stain and scan the membane to obtain an image showing the amount of protein in each lane.</li>
	<li>Destain the membrane and incubate in 5% milk in PBS for 1 hr to block.</li>
	<li>Incubate the membrane in the primary anti-HA antibody at a concentration of 1:10,000 in 5% milk in PBS either overnight at 4 &#176;C or for 4 hr at room temperature.</li>
	<li>Perform 3 washes for 5 min each in PBS with 0.1% Tween-20 detergent.</li>
	<li>Incubate the membrane in a mouse horseradish peroxidase at a concentration of 1:10,000 in 5% milk in PBS for at least 2 hr.</li>
	<li>Perform 3 washes for 5 min each in PBS with 0.1% Tween-20 detergent.</li>
	<li>Perform 1 wash for 5 min in PBS.</li>
	<li>Incubate the membrane in a chemiluminescent detection reagent for 10 min and detect using a chemiluminescent detector. Use the automatic exposure setting on the detector.</li>
</ol>

<ol>
	<li>Ovaa, H., Kessler, B. M., Rol&#233;n, U., Galardy, P. J., Ploegh, H. L., Masucci, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-+based+ubiquitin-specific+(USP)+profiling+of+virus-infected+and+malignant+human+cells.">Activity- based ubiquitin-specific (USP) profiling of virus-infected and malignant human cells.</a> Proc Natl Acad Sci USA. 101 (8), 2253-2258 (2004).</li><li>Wilkinson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitination+and+deubiquitination:+Targeting+of+proteins+for+degradation+by+the+proteasome.">Ubiquitination and deubiquitination: Targeting of proteins for degradation by the proteasome.</a> Semin Cell Dev Biol. 11 (3), 141-148 (2000).</li><li>Ziad, M. E., Wilkinson, K. D. <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+proteolysis+by+human+deubiquitinating+enzymes.">Regulation of proteolysis by human deubiquitinating enzymes.</a> Biochim Biophys Acta. 1843 (1), 114-128 (2014).</li><li>Rol&#233;n, U., 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=Activity+Profiling+of+Deubiquitinating+Enzymes+in+Cervical+Carcinoma+Biopsies+and+Cell+Lines.">Activity Profiling of Deubiquitinating Enzymes in Cervical Carcinoma Biopsies and Cell Lines.</a> Mol Carcinog. 45 (4), 260-269 (2006).</li><li>Borodovsky, 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=Chemistry-Based+Functional+Proteomics+Reveals+Novel+Members+of+the+Deubiquitinating+Enzyme+Family.">Chemistry-Based Functional Proteomics Reveals Novel Members of the Deubiquitinating Enzyme Family.</a> Chem Biol. 9 (10), 1149-1159 (2002).</li><li>Andersson, F. L., 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+effect+of+Parkinson&#8217;s-disease-associated+mutations+on+the+deubiquitinating+enzyme+UCH-L1.">The effect of Parkinson&#8217;s-disease-associated mutations on the deubiquitinating enzyme UCH-L1.</a> J Mol Biol. 407 (2), 261-272 (2011).</li><li>Poon, 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=&#946;-Amyloid+(A&#946;)+oligomers+impair+brain-derived+neurotrophic+factor+retrograde+trafficking+by+down-regulating+ubiquitin+C-terminal+hydrolase+UCH-L1.">&#946;-Amyloid (A&#946;) oligomers impair brain-derived neurotrophic factor retrograde trafficking by down-regulating ubiquitin C-terminal hydrolase UCH-L1.</a> J Biol Chem. 288 (23), 16937-16948 (2013).</li><li>Nijman, S. 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+deubiquitinating+enzyme+USP1+regulates+the+Fanconi+anemia+pathway.">The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway.</a> Mol Cell. 17 (3), 331-339 (2005).</li><li>Stevenson, L. F., Sparks, A., Allende-Vega, N., Xirodimas, D. P., Lane, D. P., Saville, M. K. <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+USP2a+regulates+the+p53+pathway+by+targeting+Mdm2.">The deubiquitinating enzyme USP2a regulates the p53 pathway by targeting Mdm2.</a> EMBO J. 26 (4), 976-986 (2007).</li><li>Coughlin, 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=Small-molecule+RA-9+inhibits+proteasome-associated+DUBs+and+ovarian+cancer+in+vitro+and+in+vivo+via+exacerbating+unfolded+protein+responses.">Small-molecule RA-9 inhibits proteasome-associated DUBs and ovarian cancer in vitro and in vivo via exacerbating unfolded protein responses.</a> Clin Cancer Res. 20 (12), 3174-3186 (2014).</li><li>Colla, 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=Endoplasmic+Reticulum+Stress+Is+Important+for+the+Manifestations+of+&#945;-Synucleinopathy+In.">Endoplasmic Reticulum Stress Is Important for the Manifestations of &#945;-Synucleinopathy In.</a> Vivo. J Neurosci. 32 (10), 3306-3320 (2012).</li></ol>

The authors have nothing to disclose.

Since ubiquitination is a fundamental cellular activity, understanding the regulatory mechanisms could be the key to unearthing the processes of disease and pathology. The use of HA tagged Ub-derived active site directed probes reported here provides an easy, but highly applicable method for studying Ub-mediated protein degradation. This method is faster and less expensive than studying each one of the DUBs individually.
In this method, the lysis of the cells is achieved via mechanical means &#8211; using the glass beads. This gentle method of lysis preserves a metabolic intracellular picture. However, a major drawback of this lysis method is its low efficiency of about 60-70%. This means a lot of cells are required to ensure lysates are concentrated enough for experiments. T75 flasks with 80 - 90% confluent cell layers should be used to produce a concentrated lysate. Furthermore, the trypsinization step of the experiment is critical because most of the attached cells must be suspended in solution. Check the flask under a microscope to make sure that a significant portion of the cells in culture are floating before transferring to the 50 ml tube. Failure to properly trypsinize adherent cells will result in a smaller pellet and by extension less lysate at a lower concentration. Lysates that are not in use should be stored at -80 &#176;C, transferred to -20 &#176;C 24 hr before use and thawed on ice the day of use. This allows the lysate to thaw evenly and reduces damage to the enzymes from rapid thawing.
To apply this method to tissues either a dounce or polytron homogenizer can be used in place of the glass beads11. The obtained lysate can be tagged with the probes immediately or stored for use at -80 &#176;C. If the dounce or polytron is used to obtain the lysate then glass beads should not be used. This leads to a reduction in the total protein concentration of the lysate. Alternatively, using primary cell culture methods the solid tissue samples could be cultured first and then lysed with the glass beads.
A major limitation of this method is that even though it allows the user to visualize the activity of the enzymes, it doesn&#8217;t give accurate information on the expression patterns of the individual DUBs. This method uses functional homology in the enzymes not structural homology. Therefore, further experiments with the individual antibodies will have to be done to explain whether the reduction in activity is a result of defects in the active site or an actual loss of the enzyme. Additionally, the lysis method used in this protocol does not break open the nuclei of the cells. DUBs in the nucleus could provide further critical information about disease processes.
Nevertheless this method is unique in its ability to provide information that cannot be obtained from any other source. Both RNA methods and the use of individual antibodies do not provide functional information about enzymes. In the future, the potential exists for the application of this method to immunohistochemical (IHC) staining. IHC staining will not only give information about the activity of the enzymes, but it will also provide insights into the location in tissue where the various DUBs are active. Also, this method could be coupled with a subcellular fractionation technique such as the one detailed by Colla et al. in 2012 to study the activity of the DUBs in membrane bound organelles12. Although this is a relatively new technique in molecular biology, there is a huge potential to expand the applications. Using active site directed molecular probes might hold the key to elucidating the etiology of many diseases.

The ubiquitin-proteasome system (UPS) serves as one of the major degradation pathways in the mammalian cell. Substrates bound for degradation in the proteasome are covalently tagged with polymers of ubiquitin (Ub)1. Before the targeted substrate enters the proteasome for degradation, the poly-ubiquitin tag must be removed. A class of enzymes known as deubiquitinating enzymes (DUBs) is responsible for the removal and recycling of ubiquitin molecules2. It has been predicted from the human genome that there are nearly a hundred DUBs working in the cell3. With such a large number of DUBs controlling Ub-mediated cellular processes, studying these enzymes presents a challenge since mRNA techniques do not give information on activity and western blotting only gives information on expression levels.
The use of influenza hemagglutinin (HA) tagged, Ub-derived active site directed probes allows for a covalent modification of the functional DUBs and therefore gives a direct visualization of the activity of these enzymes on a western blot4. The probes have a C-terminal thiol reactive group that serves as a suicide substrate for the active site cysteine residue5. With these probes, it is possible to study the activity and potential expression of many DUBs under both pathological and physiological states of the cell.
Changes in DUB activity have been implicated in a range of pathological conditions such as Parkinson&#8217;s, Alzheimer&#8217;s, anemia and various cancers6-10. This technique provides a powerful tool for the study of disease. In the present paper, we show the application of this technique in HeLa and M17 cells that have been lysed using glass beads. Additionally, we outline how to use this method in mouse spinal cord tissue samples. The information obtained from this technique can be used as a starting point for identifying therapeutic targets as well as establishing models for the study of different disease conditions. The true utility of this technique lies in its ability to provide information on multiple DUBs in a single assay.

<table border="0" cellpadding="0" cellspacing="0" style="width:615px;" width="615">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PowerGen 125 ( Homogenizer)</td>
			<td style="width:113px;">Fischer Scientific</td>
			<td style="width:119px;">14-261-02P</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vacuum-driven filters .22&#181;M&#160;</td>
			<td style="width:113px;"></td>
			<td style="width:119px;">BPV2210</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass beads, acid washed &#8804;106&#181;m (~140 U.S. sieve)</td>
			<td style="width:113px;">Sigma-aldrich</td>
			<td style="width:119px;">G4649-10G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sucrose</td>
			<td style="width:113px;">Fischer Scientific</td>
			<td style="width:119px;">S6-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DL-Dithiothreitol</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">D0632</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium Chloride</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Adenosine 5&#39;-triphosphate disodium salt hydrate</td>
			<td style="width:113px;">Sigma-aldrich</td>
			<td style="width:119px;">A26209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trizma hydrochloride&#160;</td>
			<td style="width:113px;">Sigma-aldrich</td>
			<td style="width:119px;">T3253</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:113px;">Life technologies</td>
			<td style="width:119px;">11965-092</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phosphate Buffered Saline</td>
			<td style="width:113px;">Life technologies</td>
			<td style="width:119px;">10010-023</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tissue culture flask 75cm2 w/ filter cup 250 ml 120/ cs</td>
			<td style="width:113px;">Cellstar</td>
			<td style="width:119px;">T-3001-2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">0.05% Trypsin-EDTA (1X)</td>
			<td style="width:113px;">Life technologies</td>
			<td style="width:119px;">25300-054</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HI FBS</td>
			<td style="width:113px;">Life technologies</td>
			<td style="width:119px;">16140-071</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Monoclonal Anti-HA antibody produced in mouse</td>
			<td style="width:113px;">Sigma-aldrich</td>
			<td style="width:119px;">H9658</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ubiquitin vinyl sulfone (HA-tag)</td>
			<td style="width:113px;">Enzo Life Sciences</td>
			<td style="width:119px;">BML-UW0155-0025</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laemmli&#39;s SDS-Sample Buffer (4X, reducing)</td>
			<td style="width:113px;">Boston BioProducts</td>
			<td style="width:119px;">BP-110R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pierce BCA Protein Assay Kit</td>
			<td style="width:113px;">ThermoScientific</td>
			<td align="right" style="width:119px;">23225</td>
			<td></td>
		</tr>
	</tbody>
</table>

method for measuring the activity of deubiquitinating enzymes in cell lines and tissue samples

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, techniques for studying the regulatory mechanism of this system are essential to elucidating the cellular and molecular processes of the aforementioned diseases. The use of hemagglutinin derived ubiquitin probes outlined in this paper serves as a valuable tool for the study of this system. This paper details a method that enables the user to perform assays that give a direct visualization of deubiquitinating enzyme activity. Deubiquitinating enzymes control proteasomal degradation and share functional homology at their active sites, which allows the user to investigate the activity of multiple enzymes in one assay. Lysates are obtained through gentle mechanical cell disruption and incubated with active site directed probes. Functional enzymes are tagged with the probes while inactive enzymes remain unbound. By running this assay, the user obtains information on both the activity and potential expression of multiple deubiquitinating enzymes in a fast and easy manner. The current method is significantly more efficient than using individual antibodies for the predicted one hundred deubiquitinating enzymes in the human cell.

Cultured M17 and HeLa cells were harvested using the method detailed in the protocol (3. Cell Harvesting) and mouse spinal cord tissue was obtained. The cell pellet/spinal cord tissue was placed in a tube with the lysis buffer described in the reagent preparation section. Cell pellets were lysed using glass beads (Figure 1A) and mouse spinal cord tissue samples were homogenized using the homogenizer (Figure 1B). After lysis or homogenization, the sample was then centrifuged at 5,030 x g to remove the glass beads and/or the unbroken membranes and organelles (Figure 2). Both of these methods are mechanical means of lysis to preserve enzymatic activity. The protein concentration of the cells was then determined using the BCA method. 20 &#181;g of total protein was incubated with 2 &#181;l of 1.35 &#181;M probe for 1 hr at 37 &#176;C (Figure 3). The samples were then incubated in 4x Laemmli&#8217;s sample buffer at 95 &#176;C to quench the reaction. The prepared samples were loaded on a 4-20% tris-glycine gel and run at 95 V until the ion front reached the bottom. The proteins were transferred overnight onto a PVDF membrane. The protein loading was checked using the amido black stain (Figure 4). This control step ensures that the differences in activity are due to actual cellular processes and not unequal protein loading. Equal protein amounts were used in the first experiment (Figure 4A). In the second experiment, unequal protein amounts were loaded due to the expected differences in the activity profiles of the different cell lines (Figure 4B). This was done to ensure the detection of an activity profile for the M17 cell lysates incubated with N-ethylmaleimide (NEM), which is a cysteine inhibitor and should lead to a reduced signal on the western blot. The membranes were incubated in anti-HA primary antibody, a mouse horseradish peroxidase and then detected using chemiluminescent imaging (Figure 5). Successful tagging of the DUBs using the probes results in a profile in each lane of the membrane (Figure 5A, C). The intensity of the bands at different molecular weights corresponds to the activity level of the DUB enzyme. The differences in activity levels of various DUBs across cell lines become evident when similar proteins such as UCHL1 are compared in the HeLa and M17 cells (Figure 5). The chemiluminescent visualization of the gel is critical because unlike the amido black stain, which shows the amount of protein loaded, this shows the amount of active protein that has reacted with the probes. The light image of the molecular weight standards (Figure 5B) serves as the guide for identifying the various DUBs. The mass of the probe needs to be added to the size of the protein during the analysis of the results to characterize the DUBs at the correct molecular weights.
<img alt="Figure 1" src="/files/ftp_upload/52784/52784fig1.jpg" /> 
Figure 1: Lysis Methods for Cells and Tissue. Picture showing cell lysis using glass beads in (A) versus polytron lysis in (B).
<img alt="Figure 2" src="/files/ftp_upload/52784/52784fig2.jpg" /> 
Figure 2: Centrifugation Products of Cells and Tissue. Cell lysate showing the settled glass beads and unbroken membranes and organelles at the bottom after lysis and centrifugation in (A). Tissue lysate showing the unbroken membranes and organelles at the bottom after homogenization and centrifugation in (B).
<img alt="Figure 3" src="/files/ftp_upload/52784/52784fig3highres.jpg" width="700" /> 
Figure 3: Mechanism for tagging of DUBs. A schematic showing the covalent linkage between the active site cysteine residue and the functional group of the HA-Ub-VS. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52784/52784fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52784/52784fig4highres.jpg" width="400" /> 
Figure 4: Amido black stains of the membranes. Membranes showing the amido black staining done on lysates incubated with N-ethylmaleimide (NEM), tagged with HA-Ub-VinylSulfone (VS) and run on a western blot. The samples are as follows from left to right &#8211; (A) HeLa - NEM, HeLa + NEM; (B) Mr Standards, M17 - NEM, M17 + NEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52784/52784fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52784/52784fig5highres.jpg" width="650" /> 
Figure 5: Western blots results. Membranes showing the activity levels of the various DUBs as determined by probing for Anti-HA. The samples are as follows from left to right &#8211; (A) HeLa - NEM, HeLa + NEM; (B) Mr Standards; (C) M17 - NEM, M17 + NEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52784/52784fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples at JoVE.com

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

The current protocol details a method for measuring the activity of functionally homologous deubiquitinating enzymes. Specialized probes covalently modify the enzyme and allow for detection. This method holds the potential to identify new therapeutic targets.

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, ...

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9.6K Views.  University of Minnesota. The current protocol details a method for measuring the activity of functionally homologous deubiquitinating enzymes. Specialized probes covalently modify the enzyme and allow for detection. This method holds the potential to identify new therapeutic targets.

Video: Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Measuring In Vitro ATPase Activity for Enzymatic Characterization

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary.

Measuring In Vitro ATPase Activity for Enzymatic Characterization

We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate ...

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility with high-throughput formatting. This protocol describes the use of three different methods for measuring cell proliferation in vitro including conventional hemocytometer counting chamber, a luminescence-based assay that utilizes the change in the metabolic activity of viable cells as a measure of the relative number of cells, and a multi-mode cell imager that measures cell number using a counting algorithm. Each method presents its own advantages and disadvantages for the measurement of cell proliferation, including time, cost and high-throughput compatibility. This protocol demonstrates that each method could accurately measure cell proliferation over time, and was sensitive to detect growth at differing cellular densities. Additionally, measurement of cell proliferation using a cell imager was able to provide further information such as morphology, confluence and allowed for a continual monitoring of cell proliferation over time. In conclusion, each method is capable of measuring cell proliferation, but the chosen method is user-dependent.

This protocol describes the use of three different methods for analyzing cell proliferation in breast cancer cell lines. This includes the use of conventional cell counting, luminescence-based cell viability, and cell counting through the use of a cell imager. Each offers advantages for the reproducible measurement of cell proliferation.

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility ...

Measuring Lactase Enzymatic Activity in the Teaching Lab

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...

Anaerobic Growth and Maintenance of Mammalian Cell Lines

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that the incubation in normoxic (5% CO2 in air) or hypoxic (low oxygen levels) conditions followed by an anoxic incubation (an absence of free oxygen) results in limited viability (2&#8211;3 days). A novel methodology was developed that enables an anoxic cell cultivation (for at least 17 days; the maximum time tested). The most critical aspect of this methodology is to ensure that no oxygen is introduced into the system. This is obtained by the degassing of media, and by flushing tubes, dishes, flasks, and pipettes with an anaerobic gas mixture (H2, CO2, N2) followed by permitting the materials to equilibrate to the anoxic (non-oxygen) environment prior to usage.&#160;Additional care must be exercised when acquiring photomicrographs to ensure that the micrographs obtained do not contain artifacts. In the absence of oxygen, cell morphology is significantly altered. Two distinct morphotypes are noted for all anaerobically-grown cells. The ability to grow and maintain mammalian cells in the absence of oxygen can be applied to the analysis of cell physiology, polymicrobial interactions, and the characterization of biosynthetic pathways for novel cancer drug development.

Here, we describe a new method that enables the anaerobic long-term cultivation of established cell lines. The maximum survival time that was tested is 17 days. This method is suitable for the testing of cytotoxic agents and exploring the physiology of anoxically replicating cells.

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

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Chapters in this video

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

Quantitation of γH2AX Foci in Tissue Samples

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Measuring In Vitro ATPase Activity for Enzymatic Characterization

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

Measuring Lactase Enzymatic Activity in the Teaching Lab

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

Anaerobic Growth and Maintenance of Mammalian Cell Lines

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue