The authors would like to thank Tiffany Cherry in her help in optimizing this protocol. This work was funded by National Institute of Aging (AG032320) as well as Advancing a Healthier Wisconsin.

Refer to Figure 1 for a basic schematic of the procedure beginning with the homogenate. Estimated time from preparation of cellular extracts to elution of CaM-bound proteins is about six to seven hours.
1. Tissue preparation 
<ol>
	<li>Inject organotypic hippocampal slices with a virus containing a plasmid expressing the recombinant protein of interest (in this example, green fluorescent protein (GFP)-tagged Ng) and allow tissue to express protein overnight.</li>
 <li>Approximately 12 to 18 hours after viral injection (depending on the viral expression time), prepare to collect the tissue. Add 1mL dissection buffer (10mM glucose, 4mM KCl, 26mM NaHCO3, 233mM sucrose, 5mM MgCl2, 1mM CaCl2, and 0.1% phenol-red, gassed with 5% CO2 95% O2 ) to a petri dish. Transfer cultured tissue/insert to petri dish and add 2mL dissection buffer to the insert to submerge the tissue. </li>
 <li>Collect organotypic hippocampal tissue (between 5 and 10 slices) by gently scraping the tissue free of the insert membrane using a scalpel. Specifically removing a particular region of interest (e.g. CA1) is also an option. Transfer the suspended tissue to a 1.5mL microcentrifuge tube using an inverted Pasteur pipette. </li>
 <li>Centrifuge samples at 1,500 rcf for 1 min to separate tissue from the dissection buffer. Carefully remove the supernatant by aspiration. Make sure not to disturb the pellet. </li>
 <li>For each slice used, add 30 to 60 &mu;L of homogenization buffer (150mM NaCl, 20mM Tris pH 7.5, 1mM DTT, 1&mu;g/mL leupeptin, 1&mu;g/mL chemostatin, 1&mu;g/mL antipain, 1&mu;g/mL pepstatin, and 1% Triton X-100) to the tissue and homogenize thoroughly with pestle.</li>
 <li>In order to remove cellular debris, centrifuge the remaining homogenate at 1,100 rcf for 10 min and carefully remove the supernatant by aspiration while avoiding contamination from the pellet.</li>
 <li>Take 10% of the supernatant as a sample of the input (sample 1). Store the remaining supernatant on ice during preparation of the CaM-sepharose beads for use in step 3.</li>
</ol>
Note: The tissue used here are organotypic hippocampal slices. However, one could use dissociated neurons or any other cell culturing system. In such a case, begin at step 1.4 after collecting your tissue in the appropriate manner.
2. Preparation of beads for pull-down
In handling the beads, it is important to salvage the beads and maximize the efficiency of the reactions by preventing the beads from drying on the sides of the tube. To do so, it is best to rotate your tubes on their side, allowing solution to wet the beads on the walls of the tube, immediately before centrifugation. 
<ol>
	<li>For each pull-down, pipette 400 &mu;L of suspended Calmodulin-Sepharose beads into a 2 mL microcentrifuge tube with a relatively flat-bottom to maximize surface area and interaction of your solutions with the beads during your incubations. </li>
 <li>Centrifuge beads at 21,000 rcf for 30 seconds and carefully remove the supernatant by aspiration. Make sure not to disturb the beads. </li>
 <li>To wash the beads, add 100 &mu;L of the respective homogenization buffer containing either 2 mM CaCl2 to those being used to pull-down Ca2+-CaM binding proteins and 2 mM EDTA (a known Ca2+ chelator) for beads pulling down ApoCaM binding proteins. Gently tap the tube to re-suspend beads and centrifuge at 1,500 rcf for 1 min. Carefully remove the supernatant by aspiration, making sure not to disturb the beads. </li>
</ol>
Note: For all the aspiration steps, it is recommended to use a pipette tip that has a fine opening (e.g. gel loading tips) to allow removal of solution without removing beads.
3. CaM-sepharose binding of proteins
<ol>
	<li>Split your supernatant into two conditions containing an equal volume. Depending on your condition, add the appropriate amount of CaCl2 or EDTA to your supernatant up to 2 mM concentration for each.</li>
 <li>Add supernatant from step 1.7 to beads washed in corresponding homogenization buffer. Gently tap the tube to mix.</li>
 <li>Incubate samples at 4&deg;C for 3 hrs on a shaker. Re-suspend beads every 30 min or so to increase efficiency of binding.</li>
 <li>Centrifuge tube containing samples and beads at 1,500 rcf for 3 min.</li>
 <li>Take 50&mu;L of supernatant as a sample of unbound protein (sample 2) and carefully remove the remaining supernatant by aspiration and discard.</li>
 <li>Wash beads three times as described in step 2.3 using 100&mu;L of respective homogenization buffer.</li>
</ol>
4. Elution
<ol>
	<li>Add 50&mu;L of elution buffer (50mM Tris-HCl pH 7.5, and 150mM NaCl) containing the opposite condition (10mM CaCl2 or 10mM EDTA) to the beads. For example, samples that were homogenized and bound in buffer containing Ca2+ are eluted in elution buffer containing EDTA and vice-versa.</li>
</ol>
Optional: Warming the elution buffer to 37&deg;C before adding to beads may enhance the yield.
<ol start="2">
 <li>Incubate solution with beads at room temperature for 30 min on a shaker. Mix the beads by gently tapping the tube about every 5 min. </li>
 <li>Centrifuge beads at 1,500 rcf for 3 min and carefully remove the 50&mu;L of supernatant for the bound protein (sample 3) by aspiration. Make sure not to disturb the beads. </li>
 <li>Add protein loading buffer to all samples (i.e. homogenate, unbound and bound protein as well as those still bound to the beads). </li>
</ol>
Note: To maximize elution (especially in the case of inefficient elution), add 50&mu;L of the corresponding elution buffer (e.g. adding EDTA containing buffer to beads bound in CaCl2) to the beads before heating samples to help elute any remaining bound protein and repeat steps described in 4.3 to remove remaining bound proteins.
5. SDS-PAGE and western blot 
Conduct SDS-PAGE and analyze using western blot by probing for your protein of interest and probe for a protein known to bind CaM in the opposite condition as a positive control. 
6. Representative Results
Figure 2B shows an example of a CaM-pull-down assay testing the CaM binding of GFP-tagged Ng compared to endogenous Ng. To do so, GFP-Ng was overexpressed in our organotypic hippocampal slices overnight and the tissue was homogenized. The homogenate was incubated with CaM-sepharose beads in the presence of either Ca2+ or EDTA. Homogenate input shows that the GFP-Ng was expressed in addition to endogenous Ng and Ca2+/CaM-dependent kinase II (CaMKII). As expected based on the known binding of endogenous Ng (illustrated in Fig. 2A), GFP-tagged Ng was eluted in the absence of Ca2+ (EDTA bound protein) and unbound in the presence of Ca2+ (Fig. 2B). In contrast, the control, CaMKII, was eluted only in the presence of Ca2+ (bound protein) and was unbound in its absence (EDTA). This shows that the CaM beads were functioning properly and the elutions were efficient. Most importantly, this shows that GFP-Ng binds to ApoCaM in a similar fashion to the endogenous form, suggesting that the GFP tag did not alter the function of our recombinant protein.
<img src="/files/ftp_upload/3502/3502fig1.jpg" alt="Figure 1"/> Figure 1. Outline of the CaM pull-down assay 
(A) Tissue homogenate is spun down to remove cellular debris. About 10% of the supernatant is taken as a sample of the input (1). The remaining supernatant is divided equally for the different conditions and the appropriate reagents (CaCl2 or EDTA) are added to test binding in those conditions. Each supernatant (containing either CaCl2 or EDTA) is loaded onto the respectively prepared CaM-sepharose beads and (B) incubated to allow binding. Unbound proteins are removed (2) and (C) the bound proteins (3) are eluted off of the beads using elution buffer (E.B.) containing the opposite condition as the binding. The protein composition of these three protein samples can be analyzed using SDS-PAGE and western blot analysis.
<img src="/files/ftp_upload/3502/3502fig2.jpg" alt="Figure 2"/> Figure 2. A.) Schematic of Ca2+-dependent CaM binding and elution in pull-down assay Examples are given of two types of protein that bind CaM in a Ca2+-dependent manner.
Neurogranin (Ng) represents proteins that bind apo-CaM and CaMKII represents proteins that bind to Ca2+-rich CaM. CaM is shown in its dissociated state prior to incubation with the homogenate proteins. Once incubated under conditions of high Ca2+ concentrations (2 mM) or in the presence of a Ca2+ chelator, EDTA (2 mM), the proteins will bind to CaM accordingly. Ng binds CaM in the EDTA condition as there is little to no Ca2+ present, and would be eluted off the CaM-sepharose beads in the presence of Ca2+. CaMKII, however, would bind to CaM in the presence of high amounts of Ca2+ and would dissociate once the Ca2+ was chelated.
 B.) Results from example CaM pull-down assay. This figure demonstrates the expected end result of a CaM-sepharose pull down with the samples probed for Ng and CaMKII. Both the endogenous Ng and GFP-Ng are present in the lanes of proteins bound to CaM in the presence of EDTA. No Ng is bound when samples are incubated with CaM in the presence of Ca2+, demonstrating that Ng only binds apo-CaM. Our positive control, CaMKII, on the other hand, binds to CaM only in the presence of Ca2+.

<ol>
	<li>Vincenzi, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calmodulin+in+the+regulation+of+intracellular+calcium.">Calmodulin in the regulation of intracellular calcium.</a> Proc. West Pharmacol Soc. 22, 289-294 (1979).</li><li>Cheung, W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calmodulin+plays+a+pivotal+role+in+cellular+regulation.">Calmodulin plays a pivotal role in cellular regulation.</a> Science. 207, 19-27 (1980).</li><li>Zhang, M., Tanaka, T., Ikura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-induced+conformational+transition+revealed+by+the+solution+structure+of+apo+calmodulin.">Calcium-induced conformational transition revealed by the solution structure of apo calmodulin.</a> Nat. Struct. Biol. 2, 758-767 (1995).</li><li>Alexander, K. A., Wakim, B. T., Doyle, G. S., Walsh, K. A., Storm, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+the+calmodulin-binding+domain+of+neuromodulin,+a+neurospecific+calmodulin-binding+protein.">Identification and characterization of the calmodulin-binding domain of neuromodulin, a neurospecific calmodulin-binding protein.</a> J. Biol. Chem. 263, 7544-7549 (1988).</li><li>Huang, K. P., Huang, F. L., Chen, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+7.5-kDa+protein+kinase+C+substrate+(RC3+protein,+neurogranin)+from+rat+brain.">Characterization of a 7.5-kDa protein kinase C substrate (RC3 protein, neurogranin) from rat brain.</a> Arch. Biochem. Biophys. 305, 570-580 (1993).</li><li>Bahler, M., Rhoads, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calmodulin+signaling+via+the+IQ+motif.">Calmodulin signaling via the IQ motif.</a> FEBS Lett. 513, 107-113 (2002).</li><li>Routtenberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C+activation+leading+to+protein+F1+phosphorylation+may+regulate+synaptic+plasticity+by+presynaptic+terminal+growth.">Protein kinase C activation leading to protein F1 phosphorylation may regulate synaptic plasticity by presynaptic terminal growth.</a> Behav. Neural. Biol. 44, 186-200 (1985).</li><li>Zhong, L., Cherry, T., Bies, C. E., Florence, M. A., Gerges, N. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogranin+enhances+synaptic+strength+through+its+interaction+with+calmodulin.">Neurogranin enhances synaptic strength through its interaction with calmodulin.</a> EMBO J. 28, 3027-3039 (2009).</li><li>Needham, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+and+adenosinetriphosphate+in+relation+to+muscle+contraction.">Myosin and adenosinetriphosphate in relation to muscle contraction.</a> Biochim. Biophys. Acta. 4, 42-49 (1950).</li><li>Hathaway, D. R., Adelstein, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+platelet+myosin+light+chain+kinase+requires+the+calcium-binding+protein+calmodulin+for+activity.">Human platelet myosin light chain kinase requires the calcium-binding protein calmodulin for activity.</a> Proc. Natl. Acad. Sci. U.S.A. 76, 1653-1657 (1979).</li><li>Fukunaga, K., Yamamoto, H., Matsui, K., Higashi, K., Miyamoto, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+a+Ca2+-+and+calmodulin-dependent+protein+kinase+from+rat+brain.">Purification and characterization of a Ca2+- and calmodulin-dependent protein kinase from rat brain.</a> J. Neurochem. 39, 1607-1617 (1982).</li><li>Yang, S. D., Tallant, E. A., Cheung, W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcineurin+is+a+calmodulin-dependent+protein+phosphatase.">Calcineurin is a calmodulin-dependent protein phosphatase.</a> Biochem. Biophys. Res. Commun. 106, 1419-1425 (1982).</li><li>Huestis, W. H., Nelson, M. J., Ferrell, J. E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calmodulin-dependent+spectrin+kinase+activity+in+human+erythrocytes.">Calmodulin-dependent spectrin kinase activity in human erythrocytes.</a> Prog. Clin. Biol. Res. 56, 137-155 (1981).</li><li>Yamniuk, A. P., Vogel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calmodulin's+flexibility+allows+for+promiscuity+in+its+interactions+with+target+proteins+and+peptides.">Calmodulin's flexibility allows for promiscuity in its interactions with target proteins and peptides.</a> Mol. Biotechnol. 27, 33-57 (2004).</li><li>Zhong, L., Kaleka, K. S., Gerges, N. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogranin+phosphorylation+fine-tunes+long-term+potentiation.">Neurogranin phosphorylation fine-tunes long-term potentiation.</a> Eur. J. Neurosci. 33, 244-250 (2011).</li></ol>

No conflicts of interest declared.

The provided protocol utilizes CaM-sepharose beads to investigate the Ca2+-dependence of CaM-binding proteins. Many proteins bind CaM in a Ca2+-dependent manner. These interactions are of great importance given the number of CaM-binding proteins and their critical role in many signaling pathways. In this protocol, CaM-sepharose beads are used to separate CaM-binding proteins from tissue homogenate in the presence or absence of Ca2+. The results of this simple approach will further the...

<table border="1">
 <tr>
 <td>Product</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Notes</td>
 </tr>
 <tr>
 <td>Calmodulin-Sepharose beads</td>
 <td>GE Healthcare</td>
 <td>17-0529-01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-CamKII alpha</td>
 <td>Sigma-Aldrich</td>
 <td>C6974</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-neurogranin</td>
 <td>Millipore</td>
 <td>07-425</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Gel Loading Pipet Tips</td>
 <td>Fisher</td>
 <td>02-707-138</td>
 <td>Use for aspiration of supernatants</td>
 </tr>
 <tr>
 <td>Microcentrifuge tubes (2.0 mL)</td>
 <td>Fisher</td>
 <td>05-408-146</td>
 <td>Use for all steps involving calmodulin-sepharose beads</td>
 </tr>
</table>

pull-down of calmodulin-binding proteins

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the calcium-binding protein known as calmodulin (CaM)1,2. CaM is involved at multiple levels in almost all cellular processes, including apoptosis, metabolism, smooth muscle contraction, synaptic plasticity, nerve growth, inflammation and the immune response. A number of proteins help regulate these pathways through their interaction with CaM. Many of these interactions depend on the conformation of CaM, which is distinctly different when bound to Ca2+ (Ca2+-CaM) as opposed to its Ca2+-free state (ApoCaM)3.
While most target proteins bind Ca2+-CaM, certain proteins only bind to ApoCaM. Some bind CaM through their IQ-domain, including neuromodulin4, neurogranin (Ng)5, and certain myosins6. These proteins have been shown to play important roles in presynaptic function7, postsynaptic function8, and muscle contraction9, respectively. Their ability to bind and release CaM in the absence or presence of Ca2+ is pivotal in their function. In contrast, many proteins only bind Ca2+-CaM and require this binding for their activation. Examples include myosin light chain kinase10, Ca2+/CaM-dependent kinases (CaMKs)11 and phosphatases (e.g. calcineurin)12, and spectrin kinase13, which have a variety of direct and downstream effects14.
The effects of these proteins on cellular function are often dependent on their ability to bind to CaM in a Ca2+-dependent manner. For example, we tested the relevance of Ng-CaM binding in synaptic function and how different mutations affect this binding. We generated a GFP-tagged Ng construct with specific mutations in the IQ-domain that would change the ability of Ng to bind CaM in a Ca2+-dependent manner. The study of these different mutations gave us great insight into important processes involved in synaptic function8,15. However, in such studies, it is essential to demonstrate that the mutated proteins have the expected altered binding to CaM.
Here, we present a method for testing the ability of proteins to bind to CaM in the presence or absence of Ca2+, using CaMKII and Ng as examples. This method is a form of affinity chromatography referred to as a CaM pull-down assay. It uses CaM-Sepharose beads to test proteins that bind to CaM and the influence of Ca2+ on this binding. It is considerably more time efficient and requires less protein relative to column chromatography and other assays. Altogether, this provides a valuable tool to explore Ca2+/CaM signaling and proteins that interact with CaM.

Watch this Scientific Journal Video about Pull-down of Calmodulin-binding Proteins at JoVE.com

Pull-down of Calmodulin-binding Proteins

Calmodulin (CaM) pull-down assay is an effective way to investigate the interaction of CaM with various proteins. This method uses CaM-sepharose beads for efficient and specific analysis of CaM-binding proteins. This provides an important tool to explore CaM signaling in cellular function.

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the ...

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25.3K Views.  Medical College of Wisconsin. Calmodulin (CaM) pull-down assay is an effective way to investigate the interaction of CaM with various proteins. This method uses CaM-sepharose beads for efficient and specific analysis of CaM-binding proteins. This provides an important tool to explore CaM signaling in cellular function.

Video: Pull-down of Calmodulin-binding Proteins

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

Staining Proteins in Gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. Staining of proteins with Coomassie Blue, Silver Staining, SYPRO Orange, SYPRO Ruby are demonstrated in this video.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for ...

Microcontact Printing of Proteins for Cell Biology

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. Development of microcontact printing by the Whitesides group (<a href="http://gmwgroup.harvard.edu/">http://gmwgroup.harvard.edu/</a>) in the mid-1990s revolutionalized this field by making microelectronics/microfabrication techniques accessible to laboratories focused on the life sciences. Initial implementations of this method used polydimethylsiloxane (PDMS) stamps to create patterns of functionalized chemicals on material surfaces1. Since then, a range of innovative approaches have been developed to pattern other molecules, including proteins2. This video demonstrates the basic process of creating PDMS stamps and uses them to pattern proteins, as these steps are difficult to accurately express in words. We focus on patterning the extracellular matrix protein fibronectin onto glass coverslips as a specific example of patterning.
An important component of the microcontact printing process is a topological master, from which the stamps are cast; the raised and lowered regions of the master are mirrored into the stamp and define the final pattern. Typically, a master consists of a silicon wafer coated with photoresist and then patterned by photolithography, as is done here. Creation of masters containing a specific pattern requires specialized equipment, and is best approached in consultation with a fabrication center or facility. However, almost any substrate with topology can be used as a master, such as plastic diffraction gratings (see Reagents for one example), and such serendipitous masters provide readily available, simple patterns. This protocol begins at the point of having a master in hand.

Microcontact printing is used extensively to pattern proteins and other molecules on material surfaces. We demonstrate the basic steps of this process, stamping patterns of fibronectin onto glass.

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).

Pull-down of Calmodulin-binding Proteins

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Rapid Generation of Amyloid from Native Proteins In vitro

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins