This work was funded by the Canadian Institute for Health Research (CIHR) (MOP-97774) and the Natural Sciences and Engineering Research Council of Canada (NSERC, grant nr: 480619). KS is a recipient of a KRESCENT New Investigator Award (a joint award of the Kidney Foundation of Canada, Canadian Nephrology Society and Canadian Institute of Health Research) and an Early Researcher Award from the Ontario Ministry of Research and Innovation. FW is supported by a Li Ka Shing scholarship.

1. Transformation of E. coli with the pGEX-RhoA(G17A) Construct
<ol>
 <li>Prepare LB-Agar by dissolving 2.5 g LB and 1.5 g Agar in 100 ml dH2O. Autoclave and cool to an estimated 50-55 &deg;C, which as a rule of thumb, is when the flask can be held comfortably.</li>
 <li>Prepare Ampicillin (Amp) stock by dissolving 50 mg/ ml in dH2O. Syringe filter and freeze unused aliquots. Add 100 &mu;l of Amp stock (final concentration 50 &mu;g/ml) to the LB-Agar from 1.1. Swirl to mix and pour into 10 cm bacterial dishes (15-20 ml/dish). Allow it to solidify (15-30 min.) and store unused plates inverted at 4 &deg;C for 2-3 weeks.</li>
 <li>To transform E. Coli, quickly thaw an aliquot of DH5&alpha; competent cells in an ice bath. Add 1 &mu;l of pGEXRhoA(G17A) DNA diluted to 25-50 ng/&mu;l. Flick the tube to mix and incubate on ice for 30 minutes. Heat shock at 42 &deg;C for 45 seconds and place back on ice for 2 minutes. Add 900 &mu;l SOC medium and grow for one hour at 37 &deg;C with shaking.</li>
 <li>Spread 50-100 &mu;l of the transformed bacteria on an LB-Agar-Amp plate using a bent sterile Pasteur pipette. Incubate the plate right side up in a 37 &deg;C incubator for 5 minutes and then invert and grow overnight.</li>
 <li>A single colony will be picked from the plate for preparation of the GST-tagged protein (step 2.1). For future use, wrap and store plates inverted at 4 &deg;C for about 3 weeks. In addition, bacterial stocks can be prepared for more prolonged storage by growing individual colonies in 2 ml sterile LB-Amp overnight at 37 &deg;C with shaking. Mix an aliquot with sterile 80% glycerol in a 1:1 ratio and freeze at -80 &deg;C.</li>
</ol>
2. Preparation of GST-RhoA(G17A) Beads
<ol>
 <li>Prepare LB by adding 25 g LB to 1 L dH20 and autoclaving. When cool, add 50 &mu;l Amp from stock to 50 ml LB (50 &mu;g/ml final concentration). Inoculate with a well isolated colony of transformed bacteria and grow overnight at 37 &deg;C with agitation. When at full density (OD600 &gt; 1.0) dilute with 450 ml LB-Amp and grow for an additional 30 minutes at 37 &deg;C.</li>
 <li>Prepare a 100 mM stock solution of Isopropyl B-D-thiogalactopyranoside (IPTG) by dissolving 0.238 g in 10 ml dH2O. Store in aliquots at -20 &deg;C. Induce bacteria to produce Rho protein by adding 500 &mu;l 100 mM IPTG to 500 ml culture (a final concentration of 100 &mu;M). Reduce temperature to 22-24 &deg;C and grow for ~16 h hours.</li>
 <li>Spin culture at 3600 g for 10 minutes at 4 &deg;C. If needed, the 500 ml culture can be divided into 50 ml tubes for centrifugation. Freeze pellet(s) for at least 1 hour (or preferably overnight) at -80 &deg;C.</li>
 <li>Prepare 200 ml lysis buffer containing 20 mM HEPES (0.95 g)/ pH 7.5; 150 mM NaCl (1.75 g); 5 mM MgCl2 (0.203 g); 1% TX-100 (2 ml). Prepare stock solutions of 1M DTT (1.542 g in 10 ml dH2O) and 100 mM PMSF (0.174 g/10 ml EtOH). To prepare lysis buffer +, supplement 10 ml with 1mM DTT (10 &mu;l of stock) and 1 mM PMSF (100 &mu;l of stock) and one Complete Mini Protease Inhibitor tablet.</li>
 <li>Working on ice, add 10 ml lysis buffer+ to the pellets from step 2.3. Resuspend thoroughly by gentle vortexing and pipetting. Avoid foaming. Sonicate on ice for 1 minute at setting 4 with 50% pulse. Spin the sonicated lysate at 15,000-20,000 g for 15 minutes at 4 &deg;C, and remove the clarified sonicate (supernatant) to a sterile capped 15 ml tube.</li>
 <li>Prepare the Glutathione Sepharose by gently mixing the original tube containing a 75% slurry and transfer 335 &mu;l into a 15 ml tube. Use a wide bore tip to pipette beads. Add 10 ml cold PBS, and spin 500 g for 5 minutes at 4 &deg;C. Discard the supernatant, add 1 ml lysis buffer+ to the beads and spin as for previous wash. Discard the supernatant and add lysis buffer+ to make a 50% slurry.</li>
 <li>Add 250 &mu;l of equilibrated bead slurry to the supernatant from step 2.5. Rotate at 4 &deg;C for 45 minutes.</li>
 <li>Prepare 500 ml HBS containing 20 mM HEPES (2.38 g)/pH 7.5 and 150 mM NaCl (4.38 g) in dH2O. Prepare stock solutions of 1M MgCl2 (0.952 g in 10 ml dH2O) and 1M DTT (1.542 g in 10 ml dH2O). To prepare HBS+, supplement 100 ml just before use with 5 mM MgCl2 (50 &mu;l from stock) and 1 mM DTT (100 &mu;l from stock).</li>
 <li>Spin the beads from step 2.7 at 500 g for 5 minutes at 4 &deg;C. Discard the supernatant and wash beads 2x with 10 ml lysis buffer+, and 2x with 10 ml HBS+. After the final wash, make a 50% slurry by resuspending the beads in HBS+ supplemented with BD BaculoGold protease inhibitor (20 &mu;l of 50x BD BaculoGold/ml).</li>
 <li>Dilute 10 &mu;l of the final beads preparation with 2x Laemmli sample buffer containing &beta;-mercaptoethanol. Make Bovine Serum Albumin (BSA) standards. Use a 2 mg/ml stock (0.02 g of BSA in 10 ml dH2O). Mix 10 &mu;l of stock with 10 &mu;l Laemmli (20 &mu;g final); 5 &mu;l of stock with 5 &mu;l of dH2O and 10 &mu;l Laemmli (10 &mu;g final); and 2.5 &mu;l stock with 7.5 &mu;l dH2O and 10 &mu;l Laemmli (5 &mu;g final). Boil all samples (5 min). Spin the bead sample and run supernatant with BSA standards and molecular weight markers on a 10% SDS-polyacrylamide gel.</li>
 <li>Prepare the Comassie Blue stain (0.1 g in 10 ml Acetic Acid, 40 ml Methanol and 50 ml dH2O) and the destain solution (500 ml dH2O, 400 ml methanol and 100 ml acetic acid). Store at room temperature. Stain the gel for 20-30 minutes, remove the dye (it can be reused multiple times) and rinse with destain solution twice. Continue to destain with gentle shaking for several hours until bands are clearly visible.</li>
 <li>Estimate the concentration of GST-RhoA(G17A) coupled to the beads using the BSA standards as a reference (Fig 2). Aliquot an equal volume of beads containing ~10-15 &mu;g protein into 1.5 ml micro centrifuge tubes. Store beads at 4 &deg;C to use within a day. Freeze at -80 &deg;C in HBS+/glycerol in a 3:1 ratio to use within a few days.</li>
</ol>
3. GEF Pulldown Assay with Nucleotide-free RhoA(G17A) Beads
<ol>
 <li>Grow cells in 10 cm dishes to confluence. Serum deprive for at least 3 hours and treat as required.</li>
 <li>Prepare lysis buffer+ as in step 2.4. Prepare enough lysis buffer for 700 &mu;l/dish plus some extra amount to allow for pipetting errors. Add the protease inhibitors just before use.</li>
 <li>Working on ice, remove culture medium from the dishes and wash with ice-cold HBS. Remove all the HBS and add 700 &mu;l lysis buffer+ to each dish. Swirl plates to cover all areas, scrape and collect lysates into numbered 1.5 ml tubes. Spin at 15,000 g for 1 min at 4 &deg;C. The supernatant will be used for the assay.</li>
 <li>If your cell number is equivalent in all dishes being tested, you can omit doing a protein assay, and move to step 3.5. Otherwise measure the protein concentration of each supernatant using Bio-Rad quick protein assay and equalize the supernatant for volume and concentration. The amount of total protein depends on the cell types used (typically 1-1.5 mg protein for LLC-PK1 cells).</li>
 <li>Remove 30 &mu;l of each supernatant and mix with 30 &mu;l 2x reducing Laemmli sample buffer, boil and set aside for step 3.7. Add remaining supernatants to aliquots of the GST-RhoA(G17A) beads from step 2.12. Rotate for 45 minutes at 4 &deg;C.</li>
 <li>Spin beads at 6800 g for 10 seconds at 4 &deg;C. Discard the supernatant and wash the beads 3x with lysis buffer, spinning in the same way between washes. Completely remove the final wash using a 1 cc syringe fitted with a 30 G needle and add 20 &mu;l 2x reducing Laemmli sample buffer. Boil for 5 min. Spin to pellet beads and either run the supernatant immediately (preferable) or store it at -80 &deg;C for later analysis.</li>
 <li>Run 20 &mu;l total cell lysates and all of the precipitated protein samples on the appropriate percentage SDS-polyacrylamide gel for the size of GEF you are studying. Detect your GEF of choice by Western blotting using a specific antibody.</li>
</ol>
4. Representative Results
Part 1 and 2 of the protocol describes preparation of GST-RhoA(G17A) coupled to GSH-sepharose beads and its testing by SDS-PAGE (see outline of protocol on Fig 1). A typical Coomassie stained gel is shown on Fig 2. The sample with the eluted protein should contain a single band at approximately 50 kDa (Fig 2, lane 6). The concentration of the protein can be estimated using the BSA reference samples. In the example on Fig 2, the concentration of RhoA(G17A) is estimated to be 15 &mu;g/10 &mu;l. Thus, aliquots of 10 &mu;l/tube were prepared. The typical yield in our hand is 15-20 &mu;g protein from 10 ml bacterial lysis. Part 3 of the protocol describes the affinity precipitation assay (see overview on Fig 1). A successful GEF assay detecting activation of the exchange factor GEF-H1 is shown on Fig 3. The RhoA(G17A) protein captured some GEF-H1 from the control (untreated) cell lysates, suggesting that GEF-H1 has basal activity. The amount precipitated however increases in cells treated with the inflammatory cytokine Tumor Necrosis Factor -&alpha; (TNF- &alpha;), consistent with the notion that TNF-&alpha; activates GEF-H1 5,7. Importantly, the total cell lysates show similar amounts of GEF-H1 in the control and the treated sample, suggesting that the treatment did not alter GEF-H1 levels and the input used in the assay is equal.
<img alt="Figure 1" src="/files/ftp_upload/3932/3932fig1.jpg" /> 
Figure 1. Overview of the protocol.
<img alt="Figure 2" src="/files/ftp_upload/3932/3932fig2.jpg" /> 
Figure 2. Representative result of the bead preparation protocol. A Coomassie stained gel with successful GST-RhoA(G17A) bead preparation is shown. Bead sample and BSA protein standards were separated by SDS-PAGE using a 10% acrylamide gel. To test the beads, 10 &mu;l of the final bead slurry containing GST-RhoA(G17A) is diluted 1:1 with reducing Laemmli sample buffer and boiled for 5 minutes. The beads were spun briefly and the supernatant loaded on the gel. The following samples were loaded: Lane 1: molecular weight marker (MW) (FroggaBio BLUeye prestained protein ladder); Lanes 2-4: 5, 10 and 20 &mu;g Bovine Serum Albumin (BSA); Lane 5 is empty; Lane 6: 10 &mu;l of the freshly prepared RhoA(G17A) beads. After separation is completed, the gel is stained using Coomassie Blue and subsequently destained to reveal proteins. The molecular weight of the GST-RhoA(G17A) protein is roughly 50 kDa and runs around the level of the 48 kDa marker. The concentration of the GST-Rho protein in this particular sample is estimated to be around 15 &mu;g/10 &mu;l slurry.
<img alt="Figure 3" src="/files/ftp_upload/3932/3932fig3.jpg" /> 
Figure 3. Representative GEF activation assay showing TNF-&alpha;-induced activation of GEF-H1. Confluent LLC-PK1 cells were treated with 10 ng/ml TNF-&alpha; for 5 minutes. Following treatment the cells were lysed and active GEFs were captured using GST-RhoA(G17A) bound beads. The presence of GEF-H1 in the precipitated proteins (top blot) and total cell lysate samples (bottom blot) was detected using Western blotting with an anti-GEF-H1 antibody (Cell Signaling). Please note the increased amount of precipitated GEF-H1 in TNF-&alpha;-treated versus non-treated cells relative to the equivalent inputs, indicating a successful result.
<img alt="Figure 4" src="/files/ftp_upload/3932/3932fig4.jpg" /> 
Figure 4. A "bad result". Although the GEF pulldown assay shown here resulted in some GEF-H1 captured by the beads, the amounts precipitated from control and TNF-&alpha;-treated cells are the same. Thus TNF-&alpha;, a know activator of GEF-H1 in this case did not induce activation. In this particular experiment subsequent troubleshooting suggested that the TNF-&alpha; used was not fresh enough and probably degraded.

<ol>
	<li>Jaffe, A. B., Hall, A. R. h. o. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTPases:+biochemistry+and+biology.">GTPases: biochemistry and biology.</a> Annu. Rev. Cell Dev. Biol. 21, 247-269 (2005).</li><li>Rossman, K. L., Der, C. J., Sondek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GEF+means+go:+turning+on+RHO+GTPases+with+guanine+nucleotide-exchange+factors.">GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors.</a> Nat. Rev. Mol. Cell Biol. 6, 167-180 (2005).</li><li>Arthur, W. T., Ellerbroek, S. M., Der, C. J., Burridge, K., Wennerberg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XPLN,+a+guanine+nucleotide+exchange+factor+for+RhoA+and+RhoB,+but+not+RhoC.">XPLN, a guanine nucleotide exchange factor for RhoA and RhoB, but not RhoC.</a> J. Biol. Chem. 277, 42964-42972 (2002).</li><li>Garcia-Mata, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+activated+GAPs+and+GEFs+in+cell+lysates.">Analysis of activated GAPs and GEFs in cell lysates.</a> Methods Enzymol. 406, 425-437 (2006).</li><li>Kakiashvili, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GEF-H1+Mediates+Tumor+Necrosis+Factor-{alpha}-induced+Rho+Activation+and+Myosin+Phosphorylation:+role+in+the+regulation+of+tubular+paracellular+permeability.">GEF-H1 Mediates Tumor Necrosis Factor-{alpha}-induced Rho Activation and Myosin Phosphorylation: role in the regulation of tubular paracellular permeability.</a> J. Biol. Chem. 284, 11454-11466 (2009).</li><li>Waheed, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+signal+regulated+kinase+and+GEF-H1+mediate+depolarization-induced+Rho+activation+and+paracellular+permeability+increase.">Extracellular signal regulated kinase and GEF-H1 mediate depolarization-induced Rho activation and paracellular permeability increase.</a> Am. J. Physiol. Cell Physiol. , (2010).</li><li>Kakiashvili, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidermal+growth+factor+receptor+mediates+tumor+necrosis+factor-alpha-induced+activation+of+the+ERK/GEF-H1/RhoA+pathway+in+tubular+epithelium.">The epidermal growth factor receptor mediates tumor necrosis factor-alpha-induced activation of the ERK/GEF-H1/RhoA pathway in tubular epithelium.</a> J. Biol. Chem. 286, 9268-9279 (2011).</li><li>Garcia-Mata, R., Burridge, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catching+a+GEF+by+its+tail.">Catching a GEF by its tail.</a> Trends Cell Biol. 17, 36-43 (2007).</li><li>Dubash, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+small+GTPase+RhoA+localizes+to+the+nucleus+and+is+activated+by+Net1+and+DNA+damage+signals.">The small GTPase RhoA localizes to the nucleus and is activated by Net1 and DNA damage signals.</a> PLoS One. 6, e17380-e17380 (2011).</li><li>Guilluy, G., Dubash, A. D., Garc&#237;a-Mata, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+RhoA+and+Rho+GEF+activity+in+whole+cells+and+the+cell+nucleus.">Analysis of RhoA and Rho GEF activity in whole cells and the cell nucleus.</a> Nature Protocols. , (2011).</li><li>Guilluy, C., Swaminathan, V., Garcia-Mata, R., O'Brien, E. T., Superfine, R., Burridge, 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+Rho+GEFs+LARG+and+GEF-H1+regulate+the+mechanical+response+to+force+on+integrins.">The Rho GEFs LARG and GEF-H1 regulate the mechanical response to force on integrins.</a> Nature Cell Biology. 13, 722-727 (2011).</li><li>Dubash, A. D., Wennerberg, K., Garcia-Mata, R., Menold, M. M., Arthur, W. T., Burridge, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+for+Lsc/p115+RhoGEF+and+LARG+in+regulating+RhoA+activity+downstream+of+adhesion+to+fibronectin.">A novel role for Lsc/p115 RhoGEF and LARG in regulating RhoA activity downstream of adhesion to fibronectin.</a> J. Cell Sci. 120, 3989-3998 (2007).</li><li>Arthur, W. T., Ellerbroek, S. M., Der, C. J., Burridge, K., Wennerberg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XPLN,+a+guanine+nucleotide+exchange+factor+for+RhoA+and+RhoB,+but+not+RhoC.">XPLN, a guanine nucleotide exchange factor for RhoA and RhoB, but not RhoC.</a> J. Biol. Chem. 277, 42964-42972 (2002).</li></ol>

The authors have nothing to disclose.

The method presented here is the only available non-radioactive activation assay for GEFs that can follow the active pool of GEFs in cells. The assay is similar to the precipitation assays used for following activation of small GTPases as well as GEFs against Rac and Cdc42. Those assays use different GST-tagged proteins and have slight differences from the one described here, however the basic steps are the same. Thus, this protocol can easily be adapted for other small GTPase and GEF activation assays.
The presented GEF assay was recently modified for application for nuclear fractions 9,10. With further modifications, testing of GEF activation in other subcellular compartments might also be possible.
We use the presented method to study activation of GEFs in epithelial cell lines5,6,7. With some optimization, this assay should be adequate to detect GEFs from any cell line. When adapting to a specific cell type, find the optimal cell number, lysis buffer volume, and detection method for the GEF to be tested (a good antibody for Western blotting is important). For initial setup of the assay it is advisable to use a stimulus that is known to activate the GEF of interest. When using an unknown stimulus, always use a positive control to verify that your assay is working. This assay can be used to detect activation of known GEFs by Western blotting. However, it is also adequate to identify unknown GEFs. For this, captured GEFs from control and stimulated samples should be analyzed on a Coomassie-stained gel. Bands that appear only in stimulated samples might contain activated GEFs and can be sent for identification by Mass Spectrometry (e.g. 5).
Finally, a cautionary note. The assay is based on the assumption that the posttranslational modifications rendering GEFs active are preserved after cell lysis. Indeed, this is clearly the case for a number of GEFs, and since its establishments, this assay has been used by various groups to detect activation of different GEFs, including GEF-H1, p115RhoGEF and XPLN (e.g. 5,11,12,13). Preservation of the active state however might not happen in the case of all GEFs, and therefore it is conceivable that this assay will not work for all GEFs. It also has to be noted, that many GEFs exert activity towards more than one small GTPases. Thus, when a specific GEF is studied, it is recommended to complement this assay with GEF precipitation assays for other small GTPases, as well as functional studies examining RhoA, Rac1 and Cdc42.
Critical steps in the protocol:
Colonies of transformed bacteria should be picked from fresh, properly prepared plates to ensure adequate selection by Amp, good outgrowth and yield. Transformation conditions for competent cells obtained from other sources may vary and should be consulted.
All steps of the protein preparation protocol (from step 2.3) and the assay (from step 3.2) should be performed at 4&deg;C with cooled solutions and centrifuges.
Bacterial lysis (step 2.5) should be thorough and complete in order to obtain a homogeneous suspension. When lysing the bacteria, vortex and pipette the lysate alternately, while maintaining it at 4&deg;C and ensure sonication is done on ice to prevent denaturing the protein. If using a different model of sonicator, conditions may need to be adjusted. Incubation of sonicate with the beads should always be done at 4&deg;C on a rotator to ensure sufficient binding, and care should be taken to keep the timing consistent.
GST-Rho mutants are somewhat unstable when expressed in bacteria, so it is best to use prepared beads right away or within a few days.
The precipitation assay (Part 3) is time and temperature sensitive, as active GEFs can be easily lost from the cell lysate, so steps should be performed as quickly as possible.
The following section contains some trouble-shooting tips.
No or very low amount of mutant Rho protein in the final bead preparation: This may be caused by inefficient induction, insufficient lysis of the bacteria, or a loss of the protein during the preparation process or storage. To help troubleshoot some of these possibilities, samples of bacteria can be analyzed before and after induction. If there is poor induction of the protein repeat the process using a colony from a freshly streaked plate or from re-transformed competent cells. Different IPTG concentrations and induction times should also be tested. If the lysis is insufficient (i.e. the protein remains in the pellet instead of the supernatant) varying salt and detergent concentrations in the lysis buffer can be tried. Alternate sonication times and settings should be considered, and samples before and after sonication can be checked by microscopy to determine efficiency of lysis.
No precipitated GEF, even though the GST-protein is present on the beads: This may be due to technical issues during the precipitation assay, or by a real absence of activation of the studied GEF using the stimulus applied. Always use a known stimulus as a positive control to verify that your assay works. Use the prepared beads within a few days. If the precipitation assay captures undetectable amounts of the GEF studied in all conditions, verify that your GEF is present and is well detectable in the supernatant after centrifugation that is to be used for the assay (step 3.3). Make sure all buffers and protease inhibitors are fresh, and perform all steps on ice as fast as possible. Increase the amount of input protein (e.g. by using lysates from 2 plates/sample). If the precipitation assay shows basal precipitation of your GEF, but no difference is seen between the control and stimulated samples (Fig 4), start troubleshooting by verifying that the applied stimulus worked using other known effects (e.g. by detecting activation of other signaling pathways). Consider changing the treatment conditions and/or concentrations. Rely on data from the literature reporting how your stimulus activates Rho or other signaling to predict likely times and concentrations. When optimizing treatment time, use both short and long time points, as GEF activation might be best detectable at a time point prior to well detectable Rho activation. Finally, the same stimulus could result in a variable degree of activation due to a change in cell responsiveness caused by passage number, cell confluency, etc.

<table border="1">
 <tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>pGEX-RhoA(G17A) </td>
 <td>Addgene</td>
 <td>Will be available soon</td>
 <td>Generated in the lab of Dr. Keith Burridge (University of North Carolina) (ref 3) </td>
 </tr>
 <tr>
 <td>DH5&alpha;</td>
 <td>Invitrogen</td>
 <td>18258-012</td>
 <td>Store -80&deg;C</td>
 </tr>
 <tr>
 <td>SOC medium </td>
 <td>BioShop</td>
 <td>SOC001.10</td>
 <td></td>
 </tr>
 <tr>
 <td>LB</td>
 <td>BioShop</td>
 <td>LBL407.1</td>
 <td>Keep sterile</td>
 </tr>
 <tr>
 <td>Glycerol</td>
 <td>BioShop</td>
 <td>GLY002.1</td>
 <td></td>
 </tr>
 <tr>
 <td>Ampicillin</td>
 <td>BioShop</td>
 <td>AMP201.25</td>
 <td>Store stock at -20&deg;C</td>
 </tr>
 <tr>
 <td>Agar</td>
 <td>BioShop</td>
 <td>AGR001.500</td>
 <td></td>
 </tr>
 <tr>
 <td>IPTG ( Isopropyl B-D-thiogalacto-pyranoside)</td>
 <td>Calbio-chem</td>
 <td>420322</td>
 <td>Store stock solution at -20&deg;C</td>
 </tr>
 <tr>
 <td>Glutathione Sepharose beads</td>
 <td>GE Healthcare</td>
 <td>17-0756-01</td>
 <td></td>
 </tr>
 <tr>
 <td>PBS 10x</td>
 <td>SIGMA</td>
 <td>D1408</td>
 <td></td>
 </tr>
 <tr>
 <td>Hepes</td>
 <td>SIGMA</td>
 <td>H4034</td>
 <td></td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>BioShop</td>
 <td>SOD001.10</td>
 <td></td>
 </tr>
 <tr>
 <td>MgCl2</td>
 <td>SIGMA</td>
 <td>M-9272</td>
 <td>Add just before use</td>
 </tr>
 <tr>
 <td>TX-100</td>
 <td>SIGMA-ALDRICH</td>
 <td>X100</td>
 <td></td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>OmniPur EMD</td>
 <td>3860</td>
 <td>Add just before use</td>
 </tr>
 <tr>
 <td>PMSF</td>
 <td>SIGMA</td>
 <td>P-7626</td>
 <td>Add just before use</td>
 </tr>
 <tr>
 <td>Protease Inhibitor 50x</td>
 <td>BD Baculo-Gold</td>
 <td>51-21426Z</td>
 <td>Add just before use</td>
 </tr>
 <tr>
 <td>Complete Mini, EDTA-free 10x</td>
 <td>Roche</td>
 <td>11 836 170 001</td>
 <td>Add just before use</td>
 </tr>
 <tr>
 <td>Laemmli Sample Buffer 2x</td>
 <td>Bio-Rad</td>
 <td>161-0737</td>
 <td></td>
 </tr>
 <tr>
 <td>&beta;-mercaptoethanol</td>
 <td>SIGMA</td>
 <td>M7154</td>
 <td>Add just before use</td>
 </tr>
 <tr>
 <td>Coomassie Brilliant Blue</td>
 <td>Bio-Rad</td>
 <td>R-250</td>
 <td></td>
 </tr>
 <tr>
 <td>Acetic Acid</td>
 <td>CALEDON </td>
 <td>1000-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Methanol</td>
 <td>CALEDON</td>
 <td>6701-7</td>
 <td></td>
 </tr>
 <tr>
 <td>Bio-Rad Protein Assay</td>
 <td>Bio-Rad</td>
 <td>500-0114</td>
 <td></td>
 </tr>
 <tr>
 <td>BLUeye ladder</td>
 <td>FroggaBio</td>
 <td>PM008-0500</td>
 <td></td>
 </tr>
 </tbody>
</table>
Table 1. Reagents used.
<table border="1">
 <tbody>
 <tr>
 <td>Name of equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>RC-5B centrifuge</td>
 <td>Sorvall</td>
 <td>SS-34 Rotor</td>
 <td>Use at 4&deg;C </td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Sorvall-Thermo Scientific</td>
 <td>ST-16R</td>
 <td>Use at 4&deg;C</td>
 </tr>
 <tr>
 <td>Micro centrifuge</td>
 <td>Eppendorf</td>
 <td>5417R</td>
 <td>Use at 4&deg;C</td>
 </tr>
 <tr>
 <td>Bacterial shaker</td>
 <td>INFORS HT Ecotron</td>
 <td>AG CH-1403 Bottmingen</td>
 <td>Use at 37&deg;C or at 22&deg;C</td>
 </tr>
 <tr>
 <td>Sonicator</td>
 <td>Branson</td>
 <td>Sonifier-450</td>
 <td>Use at RT</td>
 </tr>
 <tr>
 <td>Rotator</td>
 <td>Glas-Col</td>
 <td>099A CR4012</td>
 <td>Use at 4&deg;C</td>
 </tr>
 </tbody>
</table>
Table 2. Specific equipments used.

affinity precipitation of active rho-gefs using a gst-tagged mutant rho protein (gst-rhoa(g17a)) from epithelial cell lysates

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell migration, gene expression, cell cycle progression and cell adhesion 1. Rho proteins are molecular switches that are active in GTP-bound and inactive in GDP-bound state. Their activation is mediated by a family of Guanine-nucleotide Exchange Factor (GEF) proteins. Rho-GEFs constitute a large family, with overlapping specificities 2. Although a lot of progress has been made in identifying the GEFs activated by specific signals, there are still many questions remaining regarding the pathway-specific regulation of these proteins. The number of Rho-GEFs exceeds 70, and each cell expresses more than one GEF protein. In addition, many of these proteins activate not only Rho, but other members of the family, contributing further to the complexity of the regulatory networks. Importantly, exploring how GEFs are regulated requires a method to follow the active pool of individual GEFs in cells activated by different stimuli. Here we provide a step-by-step protocol for a method used to assess and quantify the available active Rho-specific GEFs using an affinity precipitation assay. This assay was developed a few years ago in the Burridge lab 3,4 and we have used it in kidney tubular cell lines 5,6,7. The assay takes advantage of a "nucleotide free" mutant RhoA, with a high affinity for active GEFs. The mutation (G17A) renders the protein unable to bind GDP or GTP and this state mimics the intermediate state that is bound to the GEF. A GST-tagged version of this mutant protein is expressed and purified from E. coli, bound to glutathione sepharose beads and used to precipitate active GEFs from lysates of untreated and stimulated cells. As most GEFs are activated via posttranslational modifications or release from inhibitory bindings, their active state is preserved in cell lysates, and they can be detected by this assay8. Captured proteins can be probed for known GEFs by detection with specific antibodies using Western blotting, or analyzed by Mass Spectrometry to identify unknown GEFs activated by certain stimuli.

Watch this Scientific Journal Video about Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein GST-RhoAG17A from Epithelial Cell Lysates at JoVE.com

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein GST-RhoAG17A from Epithelial Cell Lysates

The method presented here describes an assay to follow activation of RhoA specific GDP/GTP Exchange Factors (GEFs) in cultured cells by making use of a mutant RhoA GST fusion protein that has high affinity for activated GEFs. GEFs are precipitated from cell lysates, detected by Western blotting and quantified by densitometry.

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell ...

affinity-precipitation-active-rho-gefs-using-gst-tagged-mutant-rho

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JoVE Journal

Biology

16.5K Views.  St. Michael's Hospital. The method presented here describes an assay to follow activation of RhoA specific GDP/GTP Exchange Factors (GEFs) in cultured cells by making use of a mutant RhoA GST fusion protein that has high affinity for activated GEFs. GEFs are precipitated from cell lysates, detected by Western blotting and quantified by densitometry.

Video: Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein GST-RhoAG17A from Epithelial Cell Lysates

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency

De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro positive selection assay to measure in vivo mutations induced in a transgenic &#955;gt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.

De novo mutations in the male germline may contribute to adverse health outcomes in subsequent generations. Here we describe a protocol for the use of a transgenic rodent model for quantifying mutations in male germ cells induced by environmental agents.

De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for ...