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 c , which as a rule of thumb , is when the flask can be held comfortably . syringe filter and freeze unused aliquots . add 100 l of amp stock ( final concentration 50 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 c for 2 - 3 weeks . to transform e. coli , quickly thaw an aliquot of dh5 competent cells in an ice bath . flick the tube to mix and incubate on ice for 30 minutes . heat shock at 42 c for 45 seconds and place back on ice for 2 minutes . add 900 l soc medium and grow for one hour at 37 c with shaking . spread 50 - 100 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 c incubator for 5 minutes and then invert and grow overnight . a single colony will be picked from the plate for preparation of the gst - tagged protein ( step 2.1 ) . for future use , in addition , bacterial stocks can be prepared for more prolonged storage by growing individual colonies in 2 ml sterile lb - amp overnight at 37 c with shaking . mix an aliquot with sterile 80% glycerol in a 1:1 ratio and freeze at -80 c . when cool , add 50 l amp from stock to 50 ml lb ( 50 g / ml final concentration ) . inoculate with a well isolated colony of transformed bacteria and grow overnight at 37 c with agitation . when at full density ( od600 > 1.0 ) dilute with 450 ml lb - amp and grow for an additional 30 minutes at 37 c . prepare a 100 mm stock solution of isopropyl b - d - thiogalactopyranoside ( iptg ) by dissolving 0.238 g in 10 ml dh2o . induce bacteria to produce rho protein by adding 500 l 100 mm iptg to 500 ml culture ( a final concentration of 100 m ) . 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 c . 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 1 m 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 1 mm dtt ( 10 l of stock ) and 1 mm pmsf ( 100 l of stock ) and one complete mini protease inhibitor tablet . working on ice , spin the sonicated lysate at 15,000 - 20,000 g for 15 minutes at 4 c , and remove the clarified sonicate ( supernatant ) to a sterile capped 15 ml tube . prepare the glutathione sepharose by gently mixing the original tube containing a 75% slurry and transfer 335 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 c . discard the supernatant , add 1 ml lysis buffer+ to the beads and spin as for previous wash . 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 1 m mgcl2 ( 0.952 g in 10 ml dh2o ) and 1 m dtt ( 1.542 g in 10 ml dh2o ) . to prepare hbs+ , supplement 100 ml just before use with 5 mm mgcl2 ( 50 l from stock ) and 1 mm dtt ( 100 l from stock ) . spin the beads from step 2.7 at 500 g for 5 minutes at 4 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 l of 50x bd baculogold / ml ) . dilute 10 l of the final beads preparation with 2x laemmli sample buffer containing -mercaptoethanol . make bovine serum albumin ( bsa ) standards . use a 2 mg / ml stock ( 0.02 g of bsa in 10 ml dh2o ) . mix 10 l of stock with 10 l laemmli ( 20 g final ) ; 5 l of stock with 5 l of dh2o and 10 l laemmli ( 10 g final ) ; and 2.5 l stock with 7.5 l dh2o and 10 l laemmli ( 5 g final ) . spin the bead sample and run supernatant with bsa standards and molecular weight markers on a 10% sds - polyacrylamide gel . 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 . 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 g protein into 1.5 ml micro centrifuge tubes . freeze at -80 c in hbs+/glycerol in a 3:1 ratio to use within a few days . serum deprive for at least 3 hours and treat as required . prepare lysis buffer+ as in step 2.4 . prepare enough lysis buffer for 700 l / dish plus some extra amount to allow for pipetting errors . working on ice , remove culture medium from the dishes and wash with ice - cold hbs . swirl plates to cover all areas , scrape and collect lysates into numbered 1.5 ml tubes . 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 ) . remove 30 l of each supernatant and mix with 30 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 . 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 l 2x reducing laemmli sample buffer . spin to pellet beads and either run the supernatant immediately ( preferable ) or store it at -80 c for later analysis . run 20 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 . 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 ) . 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 ) the typical yield in our hand is 15 - 20 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 - ( tnf-  ) , consistent with the notion that tnf- activates gef - h1 . 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 . 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 l of the final bead slurry containing gst - rhoa(g17a ) the following samples were loaded : lane 1 : molecular weight marker ( mw ) ( froggabio blueye prestained protein ladder ) ; lanes 2 - 4 : 5 , 10 and 20 g bovine serum albumin ( bsa ) ; lane 5 is empty ; lane 6 : 10 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 g/10 l slurry . confluent llc - pk1 cells were treated with 10 ng / ml tnf- 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--treated versus non - treated cells relative to the equivalent inputs , indicating a successful result . although the gef pulldown assay shown here resulted in some gef - h1 captured by the beads , the amounts precipitated from control and tnf--treated cells are the same . thus tnf- , a know activator of gef - h1 in this case did not induce activation . in this particular experiment subsequent troubleshooting suggested that the tnf- used was not fresh enough and probably degraded . 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 c , which as a rule of thumb , is when the flask can be held comfortably . syringe filter and freeze unused aliquots . add 100 l of amp stock ( final concentration 50 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 c for 2 - 3 weeks . to transform e. coli , quickly thaw an aliquot of dh5 competent cells in an ice bath . flick the tube to mix and incubate on ice for 30 minutes . heat shock at 42 c for 45 seconds and place back on ice for 2 minutes . add 900 l soc medium and grow for one hour at 37 c with shaking . spread 50 - 100 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 c incubator for 5 minutes and then invert and grow overnight . a single colony will be picked from the plate for preparation of the gst - tagged protein ( step 2.1 ) . for future use , in addition , bacterial stocks can be prepared for more prolonged storage by growing individual colonies in 2 ml sterile lb - amp overnight at 37 c with shaking . mix an aliquot with sterile 80% glycerol in a 1:1 ratio and freeze at -80 c . prepare lb by adding 25 g lb to 1 l dh20 and autoclaving . when cool , add 50 l amp from stock to 50 ml lb ( 50 g / ml final concentration ) . inoculate with a well isolated colony of transformed bacteria and grow overnight at 37 c with agitation . when at full density ( od600 > 1.0 ) dilute with 450 ml lb - amp and grow for an additional 30 minutes at 37 c . prepare a 100 mm stock solution of isopropyl b - d - thiogalactopyranoside ( iptg ) by dissolving 0.238 g in 10 ml dh2o . induce bacteria to produce rho protein by adding 500 l 100 mm iptg to 500 ml culture ( a final concentration of 100 m ) . 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 c . 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 1 m 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 1 mm dtt ( 10 l of stock ) and 1 mm pmsf ( 100 l of stock ) and one complete mini protease inhibitor tablet . working on ice , spin the sonicated lysate at 15,000 - 20,000 g for 15 minutes at 4 c , and remove the clarified sonicate ( supernatant ) to a sterile capped 15 ml tube . prepare the glutathione sepharose by gently mixing the original tube containing a 75% slurry and transfer 335 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 c . discard the supernatant , 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 1 m mgcl2 ( 0.952 g in 10 ml dh2o ) and 1 m dtt ( 1.542 g in 10 ml dh2o ) . to prepare hbs+ , supplement 100 ml just before use with 5 mm mgcl2 ( 50 l from stock ) and 1 mm dtt ( 100 l from stock ) . spin the beads from step 2.7 at 500 g for 5 minutes at 4 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 l of 50x bd baculogold / ml ) . dilute 10 l of the final beads preparation with 2x laemmli sample buffer containing -mercaptoethanol . make bovine serum albumin ( bsa ) standards . use a 2 mg / ml stock ( 0.02 g of bsa in 10 ml dh2o ) . mix 10 l of stock with 10 l laemmli ( 20 g final ) ; 5 l of stock with 5 l of dh2o and 10 l laemmli ( 10 g final ) ; and 2.5 l stock with 7.5 l dh2o and 10 l laemmli ( 5 g final ) . spin the bead sample and run supernatant with bsa standards and molecular weight markers on a 10% sds - polyacrylamide gel . 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 . 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 g protein into 1.5 ml micro centrifuge tubes . freeze at -80 c in hbs+/glycerol in a 3:1 ratio to use within a few days . serum deprive for at least 3 hours and treat as required . prepare lysis buffer+ as in step 2.4 . prepare enough lysis buffer for 700 l / dish plus some extra amount to allow for pipetting errors . working on ice , remove culture medium from the dishes and wash with ice - cold hbs . swirl plates to cover all areas , scrape and collect lysates into numbered 1.5 ml tubes . 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 ) . remove 30 l of each supernatant and mix with 30 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 . 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 l 2x reducing laemmli sample buffer . spin to pellet beads and either run the supernatant immediately ( preferable ) or store it at -80 c for later analysis . run 20 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 . 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 ) . 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 ) the typical yield in our hand is 15 - 20 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 - ( tnf-  ) , consistent with the notion that tnf- activates gef - h1 . 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 . 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 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 following samples were loaded : lane 1 : molecular weight marker ( mw ) ( froggabio blueye prestained protein ladder ) ; lanes 2 - 4 : 5 , 10 and 20 g bovine serum albumin ( bsa ) ; lane 5 is empty ; lane 6 : 10 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 g/10 l slurry . confluent llc - pk1 cells were treated with 10 ng / ml tnf- 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--treated versus non - treated cells relative to the equivalent inputs , indicating a successful result . although the gef pulldown assay shown here resulted in some gef - h1 captured by the beads , the amounts precipitated from control and tnf--treated cells are the same . thus tnf- , a know activator of gef - h1 in this case did not induce activation . in this particular experiment subsequent troubleshooting 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 . with further modifications , testing of gef activation in other subcellular compartments we use the presented method to study activation of gefs in epithelial cell lines . with some optimization 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 . 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. ) . 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. ) . 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 . 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 4c 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 4c and ensure sonication is done on ice to prevent denaturing the protein . incubation of sonicate with the beads should always be done at 4c 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 . 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 . 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 . 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 ) . 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 . 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 4c 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 4c and ensure sonication is done on ice to prevent denaturing the protein . incubation of sonicate with the beads should always be done at 4c 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 . 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 . 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 ) . 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 .
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 .