This work was funded by National Institutes of Health grant GM086822, Hope Heart Institute Basic Sciences award, and the M. J. Murdock Charitable Trust College Science Research Program. Select electrophoresis reagents and image analysis software were generously provided by Bio-Rad.

1. Preparation of cell cytosol containing functional GR
<ol>
 <li>Technical note: All buffers should be refrigerated and each step of this protocol, including centrifugations and incubation, should be performed on ice or at 4 &deg;C, unless otherwise noted. The low temperature is essential to prevent degradation of the GR and protein complexes.</li>
 <li>Using a refrigerated centrifuge, obtain a &tilde;1-5 ml pellet of cells that expresses a high concentration of functional GR. Examples of cell sources rich in GR include mouse fibroblast L929 cells, which express a high concentration of endogenous GR, and Sf9 cells that have been infected with recombinant GR baculovirus (e.g. the p2Bac-mGR baculovirus 12). Approximately 15-20 ml of packed cells is obtained per liter of Sf9 baculovirus cell culture. Cells should be growing exponentially prior to harvesting. Centrifuge the cell suspension at 5,000 &times; g for 5 min. </li>
 <li>Wash the cell pellet three times with 15 ml of Hanks' buffered saline solution. Between washes, gently resuspend the pellet, followed by centrifuging at 5,000 &times; g for 5 min. Following the last wash, completely remove the supernatant.</li>
 <li>Prepare 7.5 ml of a homogenization buffer containing HEM buffer (10 mM HEPES, 1 mM EDTA, 20 mM sodium molybdate, pH 7.4), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 3 ground tablets of Complete-Mini protease inhibitor. The PMSF and Complete-Mini tablets prevent proteolysis from occurring during the subsequent steps. Complete-Mini tablets can be easily ground to a fine powder with a mortar and pestle. Add 1.5 volumes of homogenization buffer to the cell pellet.</li>
 <li>Rupture the cells by Dounce homogenization (50 strokes) or freeze/thaw (3 cycles) technique. Dounce homogenization may maintain improved endogenous GR&bull;hsp90 protein complexes and is faster to complete. Freeze/thaw provides researcher an opportunity to suspend the protocol at this step and continue at a later time. Dounce homogenization should be performed with the homogenizer in an ice bucket to prevent protein degradation. Freeze/thaw can be accomplished by freezing the centrifuge tube containing the cell pellet-homogenization buffer mixture with liquid nitrogen, dry ice, or -20 &deg;C incubation, followed by thawing in a 30 &deg;C water bath.</li>
 <li>Separate the GR-containing cytosol from the ruptured cell particulate matter by ultracentrifugation. Transfer the homogenate to durable ultracentrifuge tubes, balance tubes by mass in pairs, and centrifuge at 100,000 &times; g for 30 minutes.</li>
 <li>Remove the cytosol (supernatant) and store at -20 &deg;C in 500 &mu;l aliquots in 1.5 ml microcentrifuge tubes for long-term storage. To retain maximum functional GR activity, flash-freeze the aliquots in liquid nitrogen or incubate for 30 seconds in a methanol-dry ice bath prior to storage. Take caution to avoid direct skin contact with the cooling reagent. </li>
</ol>
2. Immunoadsorption of GR from cell cytosol
<ol>
<li>Technical note: Figure 2 provides a schematic overview of Steps 2-5 of this protocol.</li>
<li>Prepare an immunoadsorption mixture containing a monoclonal immunoglobulin G (IgG) antibody raised against the GR to immunoadsorb the receptor prepared in Step 1. In a 1.5 ml microcentrifuge tube, combine 100 &mu;l of thawed GR-containing cytosol, 200 &mu;l TEGM buffer (8 mM TES, 4 mM EDTA, 50 mM NaCl, 20 mM molybdate, 10% (v/v) glycerol, pH 7.6), 100 &mu;l of a 20% protein A-Sepharose (PAS) slurry (v/v, prepared in TEGM buffer), and 5 &mu;g of anti-GR monoclonal IgG antibody (e.g. BuGR2). Molybdate is included in the TEGM buffer in order to help stabilize the GR-hsp90 heterocomplex, which may be useful in order to assay endogenous heterocomplex steroid binding activity 13. 
 <ol>
 <li>Anti-GR antibody is commercially available as purified protein. Alternatively, it may be obtained by adding 10 &mu;l of anti-GR IgG-containing ascites to the immunoadsorption mixture. Ascites is produced by mice injected with anti-GR hybridoma cells (e.g. FiGR hybridoma cells 14), and the approximate antibody concentration obtained from ascites and used in these experiments was 0.5 &mu;g/&mu;l, as measured by Bradford assay. </li>
 <li>Prepare a negative control sample using GR-containing cytosol, TEGM buffer, and PAS (as described above), and 5 &mu;g of an IgG that does not recognize the GR, hsp90, or other molecular chaperone proteins (referred to as a "nonimmune" antibody).</li>
 <li>Due to the relatively large PAS bead size, use a cut P-200 pipet tip when transferring PAS from the 20% slurry to sample tubes. Cut pipet tips are prepared by slicing 2-3 mm off of the end of commercially available P-200 tips, thus increasing the pipet tip aperture.</li>
 </ol>
</li>
<li>Place samples on a vertical rotating wheel and incubate for a minimum of 2 hours with constant rotation. Samples may be incubated for up to 8 hours without loss of activity. </li>
<li>At the conclusion of the immunoadsorption incubation, remove unbound proteins from the antibody-PAS pellet ("immunopellet") by centrifugation. Separate the supernatants from immunopellets by centrifugation using a refrigerated centrifuge programmed for 1 minute at 10,000 &times; g, and then wash twice with 1 ml TEGM buffer and vortex. Following centrifugation and between washes, discard the supernatant being cautious to not disturb the pellet. <ol><li>Remove all the supernatant at the conclusion of the second wash. In order to ensure none of the pellet is inadvertently aspirated, use a crimped P-200 pipet tip for final supernatant aspiration. Crimped pipet tips are prepared by flattening commercially available P-200 tips using forceps.</li></ol></li>
</ol>
3. Dissociation of endogenous hsp90 from GR immunopellet ("Salt stripping")
<ol>
<li>To the washed immunopellet prepared in Step 2, add 355 &mu;l TEG buffer (TEGM buffer without sodium molybdate) and 45 &mu;l of 5 M NaCl (final NaCl concentration = 0.5 M). </li>
<li>Place samples on a vertical rotating wheel and incubate for 1.5 hours with constant rotation. Samples can be incubated for up to 2 hours; however, longer incubations may lead to decreased protein recovery and lower functional activity.</li>
<li>Remove unbound proteins from the immunopellet by centrifugation. Spin samples for 1 minute at 10,000 &times; g. Wash once with 1 ml TEG buffer and once with 1 ml of 10 mM HEPES buffer, pH 7.4.</li>
<li>Remove all the supernatant at the conclusion of the second wash, being careful not to disturb the immunopellet, using a crimped P-200 pipet tip.</li>
</ol>
4. Reconstitution of GR&bull;hsp90 heterocomplex using molecular chaperones, cofactors, and ATP
<ol>
<li>Technical note: A myriad of experimental conditions can be tested by including additional proteins, cofactors, activators, or inhibitors of interest into the reconstitution mixture prepared below. Total volume of reconstitution mixtures should be &lt;120 &mu;l. </li>
<li>To each salt-stripped immunopellet prepared in Step 3, add a reconstitution mixture containing 50 &mu;l of a molecular chaperone source and 5 &mu;l of an ATP-generating system (10 mM HEPES, 50 mM ATP, 250 mM creatine phosphate, 20 mM magnesium acetate, 100 U/ml creatine phosphokinase, pH 7.4). 
<ol>
<li>Reconstitution reactions may be increased by supplementing the reconstitution mixture with 50 &mu;l HKD buffer (10 mM HEPES, 100 mM KCl, 5 mM dithiothreitol (DTT), pH 7.4) and an additional 5 &mu;l of ATP-generating system. This step may be omitted if ample reconstitution and steroid binding are observed. KCl increases hsp70 ATPase activity and DTT protects cysteine-containing proteins from oxidation 9.</li>
<li>A recommended molecular chaperone source is commercially available rabbit reticulocyte lysate. Individual molecular chaperones purified from reticulocyte lysate or recombinantly expressed (e.g. 15 &mu;g hsp90, 15 &mu;g hsp70, 0.6 &mu;g Hop, 6 &mu;g p23, and 0.125 &mu;g hsp40 in HKD buffer) may also be used. </li>
</ol></li>
<li>Incubate samples in a 30 &deg;C water bath for exactly 20 minutes. Flick tubes every 1-2 minutes in order to gently disturb the pellet and mix the reconstitution solution. </li>
<li>Add 1 ml TEGM buffer, vortex, and centrifuge at 10,000 &times; g for 1 minute. Remove supernatant and discard being careful not to disturb the pellet. Repeat for a total of three washes. Completely remove all supernatant at the conclusion of the final wash using a crimped P-200 pipet tip.</li>
</ol>
5. Analysis of reconstituted protein complexes and ligand binding activity 
<ol>
<li>Proteins in the reconstituted immunopellet can be identified by conventional denaturing polyacrylamide gel electrophoresis (SDS-PAGE) , microfluidic electrophoresis (e.g. Bio-Rad Experion), and western blotting (Fig. 3). Technical note: Excellent protocols describing SDS-PAGE 15 and western blotting 16 are currently available, courtesy of other investigators.
<ol>
<li>Add 50 &mu;l of SDS-PAGE sample buffer containing &beta;-mercaptoethanol and vortex. Specific sample buffer recipes vary. Select one based on the recommendation of the manufacturer of the electrophoresis system to be employed.</li>
<li>Incubate for 5 minutes in a boiling water bath or 100 &deg;C electronic heat block. </li>
<li>Centrifuge at 10,000 &times; g for 1 minute. The immunoadsorbing antibody, GR, and proteins complexed to GR will be dissociated from one another and released into the sample buffer supernatant. </li>
<li>Transfer the supernatant to a fresh microcentrifuge tube using a crimped tip P-200 pipet tip. Discard the pellet. The sample is now prepared for analysis using commercially available SDS-PAGE or microfluidic electrophoresis systems. Complete either technique following manufacturer's instructions.</li>
<li>Western blot analysis following SDS-PAGE allows for definitive identification of the immunoadsorbed GR and molecular chaperones complexed to the GR following the reconstitution incubation. Use monoclonal primary antibodies raised against GR (BuGR2), hsp90 (AC88), hsp70 (N27F3-4) and co-chaperone proteins to detect the major components of the GR&bull;hsp90 protein complex.</li>
</ol></li>
<li>Functional activation of the salt-stripped GR following reconstitution of the GR&bull;hsp90 protein complex can be identified by assaying steroid binding activity using a [3H]steroid (Fig. 4). For the remainder of the protocol, follow necessary precautions for safely handling tritiated samples and waste.
<ol>
<li>To the reconstituted immunopellet, add 47.5 &mu;l HEM buffer and 2.5 &mu;l of 2 &mu;M [3H]dexamethasone (final steroid concentration = 100 nM). </li>
<li>Gently mix the pellet being careful to minimize the amount of sample displaced to the wall of the microcentrifuge tube.</li>
<li>Incubate overnight on ice. It is not necessary to rotate or mix the sample tubes during this incubation.</li>
<li>Add 1 ml TEGM buffer, gently mix, and centrifuge at 10,000 &times; g for 1 minute. Discard the supernatant, being mindful it contains unbound [3H]steroid. Repeat for a total of 3 washes with TEGM buffer. </li>
<li>Completely remove all supernatant at the conclusion of the final wash using a crimped P-200 pipet tip. The pellet contains GR, hsp90 and other molecular chaperones complexed to the GR, and the [3H]steroid specifically bound to the receptor. Nonspecific binding can be calculated by including 1000-fold excess non-tritiated dexamethasone in the overnight incubation mixture. As an alternate negative control, the anti-GR immunoadsorbing antibody added in Step 2.2 may be replaced with a non-GR-immunoadsorbing antibody (NI).</li>
<li>Suspend the washed immunopellets in 200 &mu;l TEGM buffer and transfer to 10 ml scintillation vials using a cut P-200 pipet tip. </li>
<li>Add 4.8 ml scintillation cocktail to the vials and vortex. </li>
<li>The amount of [3H]steroid binding to the reconstituted GR&bull;hsp90 protein complex can be assayed using liquid scintillation spectrometry. </li>
</ol></li>
</ol>
6. Representative Results:
 Representative SDS-PAGE and western blot data are presented in Figure 3. The endogenous GR&bull;hsp90 heterocomplex is immunoadsorbed from cell cytosol using anti-GR antibody and individual proteins that co-immunoadsorb with the GR are visualized by Coomassie staining (Fig. 3A). Confirmation of protein identity of any component of the immunopellet at any step in the reconstitution assay can be made by SDS-PAGE calculation of molecular weight and western blotting using monoclonal antibodies raised against the protein of interest. 
 Reconstituted GR&bull;hsp90 heterocomplexes are presented using Experion microfluidic electrophoresis data (Fig. 3C and 3D) and western blot analysis (Fig. 3B). The specificity of the GR immunoadsorption is confirmed by replacing the immunoadsorbing anti-GR antibody with a nonimmune antibody (Fig. 3C, NI IgG lanes). Even when excessive amounts of nonimmune antibody are used, no GR or molecular chaperones are immunoprecipitated (cf. in Fig. 3C, the GR, hsp90, and hsp70 protein bands present in the anti-GR immunoadsorption lanes with the lack of band observed in the NI IgG lanes). Likewise, it is possible to confirm the dissociation of hsp90 and other chaperone proteins from the salt-stripped GR by western blot analysis (Fig. 3B) of the stripped and reconstituted immunopellets. The heavy chain of the immunoadsorbing antibody (HC) is detectable by both Coomassie stain and western blotting, and it serves to confirm equal sized immunopellets are being visualized in each lane. 
 
 Steroid binding activity of the immunoadsorbed GR can be assayed by for all the conditions tested, and representative steroid binding data are presented in Figure 4. Endogenous GR&bull;hsp90 heterocomplexes are immunoadsorbed, salt-stripped, and reconstituted. Using either reticulocyte lysate or purified proteins, steroid binding activity of the reconstituted GR&bull;hsp90 heterocomplex typically returns to 75-100% of the endogenous GR&bull;hsp90 binding activity level. Similar to the electrophoresis data, a nonimmune antibody (NI) may be used in place of the anti-GR antibody (I) in order to serve as a negative control and as a measure of nonspecific [3H]steroid binding.

<img src="/files/ftp_upload/3059/3059fig1.jpg" alt="Figure 1"/> Figure 1. Model of GR&bull;hsp90 heterocomplex assembly and GR steroid binding cleft opening. The hsp90/hsp70-based chaperone machinery converts the GR ligand binding domain from a folded conformation in which the steroid binding cleft is closed and not accessible to hormone to an open cleft conformation that can be accessed (illustrated by the heterocyclic steroid icon). The chaperone machinery is preassembled in the cell and will spontaneously form when purified hsp90, hsp70, and Hop are mixed in solution (Reaction 1). Hop contains a 34 amino acid tetratricopeptide repeat (TPR) domain (illustrated as a dark crescent), and is later replaced during the heterocomplex maturation process by a TPR domain-containing immunophilin protein. The machinery opens the steroid binding cleft in an ATP- and K+-dependent manner, after which Hop and most of the hsp70 and hsp40 ultimately leave the complex (illustrated as a dashed icon). Mechanistically, hsp70 interacts with the GR prior to hsp90 and primes the receptor for hsp90 and subsequent cleft opening. Experimentally, steroid binding and GR cleft opening are increased if hsp90 and hsp70 are simultaneously present. The heterocomplex is stabilized by entry of the hsp90 co-chaperone p23, which maintains hsp90 in an ATP-bound conformation. After the exit of Hop, a high molecular weight immunophilin (IMM) can bind hsp90, forming the final heterocomplex as it is recovered from cells. Adapted from Pratt and Toft 1. 
<img src="/files/ftp_upload/3059/3059fig2.jpg" alt="Figure 2"/> Figure 2. Schematic representation of Steps 2-5 of this protocol. The GR is immunoadsorbed from cell cytosol using a monoclonal antibody raised against the GR (FiGR) bound to a pellet of protein A-Sepharose. Hsp90 and other chaperone proteins present in the endogenous GR&bull;hsp90 heterocomplex are co-immunoadsorbed with the GR, and the GR is able to bind to steroid. Hsp90 is dissociated from GR using a mild salt treatment, referred to as salt-stripping (Str). The steroid binding cleft of the salt-stripped GR closes, making it unable to bind to steroid. Intermediate GR&bull;hsp90 heterocomplexes containing hsp70, hsp40, and Hop, which are capable of binding steroid, can be reconstituted (Recon) by incubating the salt-stripped GR with purified proteins or reticulocyte lysate (Retic lysate) and cofactors. The steroid binding activity and immunopellet protein content of each step can be identified by overnight [3H]steroid incubation and western blotting, respectively. 
<img src="/files/ftp_upload/3059/3059fig3.jpg" alt="Figure 3"/> Figure 3. Electrophoretic protein analysis of immunoadsorbed complexes. A, SDS-PAGE of reconstituted GR&bull;hsp90 heterocomplex imaged using conventional electrophoresis system followed by electrotransfer and Coomassie staining. In addition to GR, hsp90, and hsp70, the heavy chain (HC) and light chain (LC) of the antibodies are visualized. Migration of molecular weight markers (expressed in kDa) are indicated on the left. B, Western blot of immunopellets containing salt-stripped GR (Str) and reconstituted GR&bull;hsp90 heterocomplex (Recon). C, virtual gel of reconstituted GR&bull;hsp90 heterocomplex generated using an Experion microfluidic electrophoresis system. Two different concentrations of a nonimmune (NI IgG) and immune (anti-GR) immunoadsorbing antibodies are included. NI IgGs serve as negative controls. D, electropherogram of reconstituted GR&bull;hsp90 heterocomplex obtained from Experion microfluidic electrophoresis sample shown in the third lane of panel B. Advantages of microfluidic electrophoresis include the requirement for smaller sample volumes, decreased liquid handling and post-run cleanup, improved quantification, and substantially shorter sample runs. 
<img src="/files/ftp_upload/3059/3059fig4.jpg" alt="Figure 4"/> Figure 4. Steroid binding activity of immunopellets following immunoadsorption of endogenous GR (Enodg), salt-stripped GR (Str), and reconstituted GR&bull;hsp90 heterocomplex (Recon). In addition to immunoadsorbing the GR with a monoclonal anti-GR immunoadsorbing antibody (I), negative control samples for each condition were prepared using a nonimmune immunoglobulin (NI).

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O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functioning+of+the+Hsp90+machine+in+chaperoning+checkpoint+kinase+I+(Chk1)+and+the+progesterone+receptor+(PR).">Functioning of the Hsp90 machine in chaperoning checkpoint kinase I (Chk1) and the progesterone receptor (PR).</a> Cell Stress Chaperones. 12, 353-363 (2007).</li><li>Cintron, N. S., Toft, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+requirements+for+Hsp40+and+Hsp70+in+the+Hsp90+chaperone+pathway.">Defining the requirements for Hsp40 and Hsp70 in the Hsp90 chaperone pathway.</a> J. Biol. Chem. 281, 26235-26244 (2006).</li></ol>

Select electrophoresis reagents and image analysis software were provided by Bio-Rad.

The assay described above can be adapted to test a myriad of conditions affecting the chaperone actions of the hsp90/hsp70-based chaperone machinery as well as GR steroid binding. It is a modification of previously reported methods 4,8,9,17 and is designed to take advantage of recent advances in electrophoresis technology and be accessible to a broader research community. Additional cofactors or proteins of interest may be added to the GR&bull;hsp90 heterocomplex reconstitution mixture, described in Step 4 of...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>Sf9 insect cells </td>
			<td>Invitrogen</td>
			<td>&nbsp;</td>
			<td>for baculovirus expression of recombinant mouse GR</td>
		</tr>
		<tr>
			<td>L929 cells </td>
			<td>ATCC</td>
			<td>CRL-2173</td>
			<td>for endogenous mouse GR cytosol preparation (alternative to baculovirus expression) </td>
		</tr>
		<tr>
			<td>FiGR hybridoma cells</td>
			<td>ATCC</td>
			<td>CRL-2173</td>
			<td>for preparing ascites containing anti-GR monoclonal antibody (alternative to purified BuGR2 antibody)</td>
		</tr>
		<tr>
			<td>Complete-Mini protease inhibitor, EDTA-free </td>
			<td>Roche Applied Science</td>
			<td>11836170001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>protein A-Sepharose</td>
			<td>Sigma-Aldrich</td>
			<td>P3391</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>rabbit reticulocyte lysate </td>
			<td>Green Hectares (Oregon, WI)</td>
			<td>&nbsp;</td>
			<td>rich source of endogenous hsp90/hsp70 chaperone machinery</td>
		</tr>
		<tr>
			<td>creatine phosphokinase </td>
			<td>Sigma-Aldrich</td>
			<td>C3755</td>
			<td>for ATP-regenerating system</td>
		</tr>
		<tr>
			<td>phosphocreatine </td>
			<td>Sigma-Aldrich</td>
			<td>P7936</td>
			<td>for ATP-regenerating system</td>
		</tr>
		<tr>
			<td>anti-GR mouse monoclonal antibody (BuGR2)</td>
			<td>Pierce/Thermo Scientific</td>
			<td>MA1-510</td>
			<td>used for GR immunoadsorption and as primary antibody in western blotting</td>
		</tr>
		<tr>
			<td>anti-hsp90 mouse monoclonal antibody (AC88) </td>
			<td>Enzo Life Sciences</td>
			<td>ADI-SPA-830</td>
			<td>used as primary antibody in western blotting</td>
		</tr>
		<tr>
			<td>anti-hsp70 mouse monoclonal antibody (N27F3-4) </td>
			<td>Enzo Life Sciences</td>
			<td>ADI-SPA-820</td>
			<td>used as primary antibody in western blotting</td>
		</tr>
		<tr>
			<td>IgG from mouse serum </td>
			<td>Sigma-Aldrich</td>
			<td>038K7690</td>
			<td>used as nonimmune antibody for negative control of immunoadsorption</td>
		</tr>
		<tr>
			<td>anti-mouse IgG-peroxidase conjugate </td>
			<td>Sigma-Aldrich</td>
			<td>A4416</td>
			<td>used as secondary antibody in western blotting</td>
		</tr>
		<tr>
			<td>Experion microfluidic electrophoresis system</td>
			<td>Bio-Rad</td>
			<td>7007001</td>
			<td>alternative to conventional SDS-PAGE</td>
		</tr>
		<tr>
			<td>[1,2,4,6,7-3H] dexamethasone</td>
			<td>PerkinElmer</td>
			<td>NET1192001MC</td>
			<td>follow safety precautions</td>
		</tr>
	</tbody>
</table>

biochemical reconstitution of steroid receptor&#x2022;hsp90 protein  complexes and reactivation of ligand binding

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, including transcription factors (e.g. nuclear receptors, p53) and protein kinases (e.g. Src, Raf, Akt kinase) involved in cell cycling, tumorigenesis, apoptosis, and multiple eukaryotic signaling pathways 1,2. Of these many 'client' proteins for hsp90, the assembly of steroid receptor&bull;hsp90 complexes is the best defined (Figure 1). We present here an adaptable glucocorticoid receptor (GR) immunoprecipitation assay and in vitro GR&bull;hsp90 reconstitution method that may be readily used to probe eukaryotic hsp90 functional activity, hsp90-mediated steroid receptor ligand binding, and molecular chaperone cofactor requirements. For example, this assay can be used to test hsp90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process. 
The GR has been a particularly useful system for studying hsp90 because the receptor must be bound to hsp90 to have an open ligand binding cleft that is accessible to steroid 3. Endogenous, unliganded GR is present in the cytoplasm of mammalian cells noncovalently bound to hsp90. As found in the endogenous GR&bull;hsp90 heterocomplex, the GR ligand binding cleft is open and capable of binding steroid. If hsp90 dissociates from the GR or if its function is inhibited, the receptor is unable to bind steroid and requires reconstitution of the GR&bull;hsp90 heterocomplex before steroid binding activity is restored 4 . GR can be immunoprecipitated from cell cytosol using a monoclonal antibody, and proteins such as hsp90 complexed to the GR can be assayed by western blot. Steroid binding activity of the immunoprecipitated GR can be determined by incubating the immunopellet with [3H]steroid. 
Previous experiments have shown hsp90-mediated opening of the GR ligand binding cleft requires hsp70, a second molecular chaperone also essential for eukaryotic cell viability. Biochemical activity of hsp90 and hsp70 are catalyzed by co-chaperone proteins Hop, hsp40, and p23 5. A multiprotein chaperone machinery containing hsp90, hsp70, Hop, and hsp40 are endogenously present in eukaryotic cell cytoplasm, and reticulocyte lysate provides a chaperone-rich protein source 6. 
In the method presented, GR is immunoadsorbed from cell cytosol and stripped of the endogenous hsp90/hsp70 chaperone machinery using mild salt conditions. The salt-stripped GR is then incubated with reticulocyte lysate, ATP, and K+, which results in the reconstitution of the GR&bull;hsp90 heterocomplex and reactivation of steroid binding activity 7. This method can be utilized to test the effects of various chaperone cofactors, novel proteins, and experimental hsp90 or GR inhibitors in order to determine their functional significance on hsp90-mediated steroid binding 8-11.

Watch this Scientific Journal Video about Biochemical Reconstitution of Steroid Receptorâ€¢Hsp90 Protein  Complexes and Reactivation of Ligand Binding at JoVE.com

Biochemical Reconstitution of Steroid Receptor&amp;#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

An in vitro method for preparing functional glucocorticoid receptor (GR)&#x2022;hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications.

Biochemical Reconstitution of Steroid Receptor&#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, ...

biochemical-reconstitution-steroid-receptorhsp90-protein-complexes

Research

JoVE Journal

Biology

16.3K visualizações.  Seattle University. Um In vitro Método para preparar receptor glicocorticóide funcional (GR) • hsp90 complexos de proteína purificada a partir de proteínas e lisados ​​celulares é descrito. O método utiliza de imunoadsorção recombinante GR seguido de sal de extracção e reconstituição de proteínas complexas. A importância da co-fatores e condições de buffer são discutidos, assim como as aplicações método potencial.

Vídeo: Biochemical Reconstitution of Steroid Receptor&#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.