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 ...

Research

JoVE Journal

Biology

25.2K ビュー.  Medical College of Wisconsin. カルモジュリン（CaM）プルダウンアッセイは、様々なタンパク質とカルモジュリンの相互作用を調査するために効果的な方法です。このメソッドは、CAM -結合タンパク質の効率的かつ特定の分析のためのCaM -セファロースビーズを使用しています。これは、細胞機能のCAM信号を探索するための重要なツールが用意されています。

ビデオ: Pull-down of Calmodulin-binding Proteins

Non-Radioactive In Vitro Cardiac Myosin Light Chain Kinase Assays

Cardiac-specific myosin regulatory light chain kinase (cMLCK) regulates cardiac sarcomere structure and contractility by phosphorylating the ventricular isoform of the myosin regulatory light chain (MLC2v). MLC2v phosphorylation levels are significantly reduced in failing hearts, indicating the clinical importance of assessing the activity of cMLCK and the phosphorylation level of MLC2v to elucidate the pathogenesis of heart failure. This paper describes nonradioactive methods to assess both the activity of cMLCK and MLC2v phosphorylation levels. In vitro kinase reactions are performed using recombinant cMLCK with recombinant calmodulin and MLC2v in the presence of ATP and calcium at 25 &#176;C, which are followed by either a bioluminescent ADP detection assay or a phosphate-affinity SDS-PAGE. In the representative study, the bioluminescent ADP detection assay showed a strict linear increase of the signal at cMLCK concentrations between 1.25 nM to 25 nM. Phosphate-affinity SDS-PAGE also showed a linear increase of phosphorylated MLC2v in the same cMLCK concentration range. Next, the time-dependency of the reactions was examined at the concentration of 5 nM cMLCK. A bioluminescent ADP detection assay showed a linear increase in the signal during 90 min of the reaction. Similarly, phosphate-affinity SDS-PAGE showed a time-dependent increase of phosphorylated MLC2v. The biochemical parameters of cMLCK for MLC2v were determined by a Michaelis-Menten plot using the bioluminescent ADP detection assay. The Vmax was 1.65 &#177; 0.10 mol/min/mol kinase and the average Km was around 0.5 USA &#181;M at 25 &#176;C. Next, the activity of wild type and the dilated cardiomyopathy-associated p.Pro639Valfs*15 mutant cMLCK were measured. The bioluminescent ADP detection assay and phosphate-affinity SDS-PAGE correctly detected defects in cMLCK activity and MLC2v phosphorylation, respectively. In conclusion, a combination of the bioluminescent ADP detection assay and the phosphate-affinity SDS-PAGE is a simple, accurate, safe, low-cost, and flexible method to measure cMLCK activity and the phosphorylation level of MLC2v.

This study describes protocols for nonradiometric methods, a bioluminescent ADP detection assay and a phosphate-affinity SDS-PAGE, to determine the kinase activity of cardiac myosin light chain kinase (cMLCK) and the phosphorylation level of its substrate, myosin regulatory light chain (MLC2v).

Cardiac-specific myosin regulatory light chain kinase (cMLCK) regulates cardiac sarcomere structure and contractility by phosphorylating the ventricular ...

Biochemistry

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA)

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins of several nucleotide-derived secondary messengers (NSMs) have been systematically identified and studied in model organisms. These achievements are mainly due to the development of several new techniques including the capture compound technique and the differential radial capillary action of ligand assay (DRaCALA), which were used to systematically identify target proteins of these small molecules. This paper describes the use of the NSMs, guanosine penta- and tetraphosphates (p)ppGpp, as an example and video demonstration of the DRaCALA technique. Using DRaCALA, 9 out of 20 known and 12 new target proteins of (p)ppGpp were identified in the model organism, Escherichia coli K-12, demonstrating the power of this assay. In principle, DRaCALA could be used for studying small ligands that can be labeled by radioactive isotopes or fluorescent dyes. The critical steps, pros, and cons of DRaCALA are discussed here for further application of this technique.

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA

The Differential Radial Capillary Action of Ligand Assay (DRaCALA) can be used to identify small ligand binding proteins of an organism by using an ORFeome library.

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins ...

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble effectively in vitro. Such complexes may require co-translational assembly and the presence of molecular chaperones; either they form stable oligomers which cannot dissociate and re-associate in vitro or are unstable without a binding partner. To overcome these problems, it is possible to use a method based on the bacterial co-expression of differentially tagged proteins using a set of compatible vectors followed by the conventional pulldown techniques. The workflow is more time-efficient compared to traditional pulldown because it lacks the time-consuming steps of separate purification of interacting proteins and their following incubation. Another advantage is a higher reproducibility due to a significantly smaller number of steps and a shorter period of time in which proteins that exist within the in vitro environment are exposed to proteolysis and oxidation. The method was successfully applied for studying a number of protein-protein interactions when other in vitro techniques were found to be unsuitable. The method can be used for batch testing protein-protein interactions. Representative results are shown for studies of interactions between BTB domain and intrinsically disordered proteins, and of heterodimers of zinc-finger-associated domains.

Here, we describe a method for the bacterial co-expression of differentially tagged proteins using a set of compatible vectors, followed by the conventional pulldown techniques to study protein complexes that cannot assemble in vitro.

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble ...

Studying Protease-Activated Receptor Ligands in Endothelial Cells

Characterizing Modulators of Protease-Activated Receptors with a Calcium Mobilization Assay Using a Plate Reader

Changes in calcium concentration in cells are rapidly monitored in a high-throughput fashion with the use of intracellular, fluorescent, calcium-binding dyes and imaging instruments that can measure fluorescent emissions from up to 1,536 wells simultaneously. However, these instruments are much more expensive and can be challenging to maintain relative to widely available plate readers that scan wells individually. Described here is an optimized plate reader assay for use with an endothelial cell line (EA.hy926) to measure the protease-activated receptor (PAR)-driven activation of G&#945;q signaling and subsequent calcium mobilization using the calcium-binding dye Fluo-4. This assay has been used to characterize a range of PAR ligands, including the allosteric PAR1-targeting anti-inflammatory "parmodulin" ligands identified in the Dockendorff lab. This protocol obviates the need for an automated liquid handler and permits the medium-throughput screening of PAR ligands in 96-well plates and should be applicable to the study of other receptors that initiate calcium mobilization.

Author Spotlight: Developing Parmodulins to Target Protease-Activated Receptors for Inflammation Control

An improved protocol for a calcium mobilization assay with endothelial cells, used to identify ligands of protease-activated receptors (PARs), has been developed. The new protocol reduces total assay time by 90-120 min and yields reproducible concentration-response curves.

Changes in calcium concentration in cells are rapidly monitored in a high-throughput fashion with the use of intracellular, fluorescent, calcium-binding ...

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of intracellular calcium release are the major intracellular calcium storage organelles, the endoplasmic reticulum (ER) and the more specialized sarcoplasmic reticulum (SR) in muscle cells. The dynamic release of calcium from these organelles is mediated by the ryanodine receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R) with refilling occurring through the sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. A genetically encoded calcium sensor (GECI) called CatchER was created to monitor the rapid calcium release from the ER/SR. Here, the detailed protocols for the transfection and expression of the improved, ER/SR-targeted GECI CatchER+ in HEK293 and C2C12 cells and its application in monitoring IP3R, RyR, and SERCA pump-mediated calcium transients in HEK293 cells using fluorescence microscopy is outlined. The receptor agonist or inhibitor of choice is dispersed in the chamber solution and the intensity changes are recorded in real time. With this method, a decrease in ER calcium is seen with RyR activation with 4-chloro-m-cresol (4-cmc), the indirect activation of IP3R with adenosine triphosphate (ATP), and inhibition of the SERCA pump with cyclopiazonic acid (CPA). We also discuss protocols for determining the in situ Kd and quantifying basal [Ca2+] in C2C12 cells. In summary, these protocols, used in conjunction with CatchER+, can elicit receptor mediated calcium release from the ER with future application in studying ER/SR calcium related pathologies.

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

We present protocols for the application of our targeted genetically-encoded calcium indicator (GECI) CatchER+ for monitoring rapid calcium transients in the endoplasmic/sarcoplasmic reticulum (ER/SR) of HEK293 and skeletal muscle C2C12 cells using real-time fluorescence microscopy. A protocol for the in situ Kd measurement and calibration is also discussed.

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of ...

Determination of Tripartite Interaction between Two Monomers of a MADS-box Transcription Factor and a Calcium Sensor Protein by BiFC-FRET-FLIM Assay

Protein-protein interactions are an integral part of all biological processes in the cells as they play a crucial role in regulating, maintaining, and amending cellular functions. These interactions are involved in a wide range of phenomena such as signal transduction, pathogen response, cell-cell interactions, metabolic and developmental processes. In the case of transcription factors, these interactions may lead to oligomerization of subunits, sequestering in specific subcellular contexts such as the nucleus, cytoplasm, etc., which, in turn, might have a more profound effect on the expression of the downstream genes. Here, we demonstrate a methodology to visualize in vivo tripartite interaction using Bimolecular Fluorescence Complementation (BiFC) based F&#246;rster Resonance Energy Transfer (FRET) involving Fluorescence Lifetime Imaging (FLIM). Two of the proteins selected for this demonstration interact as BiFC partners, and their reconstituted fluorescence activity is used to assay FRET-FLIM with the third partner. Four to five-week-old growth-chamber-grown Nicotiana benthamiana plants have been used as the model plant system for this demonstration.

Here we present, a method to visualize ternary complex formation between three protein partners using fluorescent-tagged proteins by BiFC based FRET-FLIM assay. This method is valuable for studying protein-protein interaction complexes in vivo.

Protein-protein interactions are an integral part of all biological processes in the cells as they play a crucial role in regulating, maintaining, and ...

Co-Immunoprecipitation and Pull-Down Assays

Co-immunoprecipitation (CoIP) and pull-down assays are closely related methods to identify stable protein-protein interactions. These methods are related to immunoprecipitation, a method for separating a target protein bound to an antibody from unbound proteins. In CoIP, an antibody-bound protein is itself bound to another protein that does not bind with the antibody, this is followed by a separation process that preserves the protein-protein complex. The difference in pull-down assays is that affinity-tagged bait proteins replace antibodies, and affinity chromatography is used to isolate protein-protein complexes.
This video explains CoIP, pull-down assays, and their implementation in the laboratory. A step-by-step protocol for each technique is covered, including the reagents, apparatus, and instruments used to purify and analyze bound proteins. Additionally, the applications section of this video describes a procedure to study how myxovirus proteins inhibit influenza nucleoprotein, an investigation into the role of calcium ions in calmodulin via a pull-down assay, and a modified pull-down assay for characterizing transient protein interactions.
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Protein-protein interactions play a significant role in a wide variety of biological functions. The majority of protein-protein interactions and their biological effects have yet to be identified. Co-immunoprecipitation, or CoIP, and pull-down assays are two closely related methods for the identification of stable protein-protein interactions. This video will cover the principles of the two assays, their general laboratory procedures, and applications of these techniques.
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CoIP and pull-down assays are variants of immunoprecipitation, a method for selectively isolating a protein species from a complex solution. In an immunoprecipitation experiment, an antibody specific to a target protein is allowed to form an immune complex with that target in the sample. The complex is then captured on a solid support, typically protein A bound to a sepharose bead. Any proteins not captured are removed by centrifugation steps. The protein is then released from the antibody and solid support by boiling in reducing SDS-PAGE sample-loading buffer.
Co-immunoprecipitation is conducted in the same manner, except that intact protein complexes are captured onto the solid support. The antibody binds to the target protein, which, in turn, is bound to another protein that is not targeted by the antibody. As in immunoprecipitation, the protein complex is released from the antibody and solid support by boiling in reducing SDS-PAGE sample loading buffer.
Pull-down assays are similar to co-immunoprecipitation, differing only in the use of a &#34;bait&#34; protein, as opposed to an antibody. Through molecular biology techniques, this bait protein is engineered with an affinity tag, such as a series of histidine residues. These affinity tags are described in &#34;Chromatography-based Biomolecule Separations.&#34; The protein is then captured on an immobilized affinity ligand specific for the tag. The captured protein is then incubated with a sample that contains proteins that form complexes with the &#34;bait&#34;. The protein complex is released from the affinity support by washing with a solution containing a competitive analyte specific for the tag on the &#34;bait&#34; protein. Pull-down assays are useful for confirming protein interactions predicted by co-immunoprecipitation, and for discovering unknown protein interactions.
Now that the principles of co-immunoprecipitation and pull-down assays have been discussed, let&#39;s look at their laboratory procedures.
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First let&#39;s discuss co-immunoprecipitation. To a series of microfuge tubes, the following is added: PBS buffer, and a 50% solution of protein A-sepharose, a resin-protein complex that binds to the antibody. The microfuge tubes are rotated to ensure proper distribution, and then the resin is washed with additional PBS buffer. Cell lysate, containing the desired protein, and 2 &#956;g of antibody are added to the microfuge tubes, and the mixture is rotated for 1 h at 4 &#176;C. The beads are pelleted by centrifugation, the supernatant is discarded, and the beads re-washed three times with buffer to remove non-bound protein. The beads containing the antibody-protein complex are in reducing SDS-PAGE sample-loading buffer, for removal of the complex from the antibody and for analysis by SDS-PAGE and immunoblotting.
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Now we will discuss the procedure for pull-down assays: The &#34;bait&#34; protein is expressed in a plasmid with the appropriate affinity tag. After reaching log-phase growth, the cells are lysed, and then centrifuged. Suspended streptavidin-sepharose beads, which capture biotin-tagged &#34;bait&#34; protein, are pipetted into a microfuge tube. The beads are then centrifuged and the supernatant carefully removed by aspiration. The beads are then washed with buffer, centrifuged, and the supernatant removed.
Cells containing the putative &#34;prey&#34; protein, which has an affinity for the &#34;bait&#34; protein, are harvested by centrifugation. The supernatant is then added to the microfuge tube containing the resin, and incubated at 4 &#176;C for 3 h on a shaker. The resin is then centrifuged, the supernatant removed, and the resin washed to remove non-bound protein. Elution buffer is added to the resin, and the mixture is incubated at room temperature for 30 min on a shaker. The resin is then centrifuged, and the supernatant containing the desired complex is analyzed by immunoblotting.
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Now that we&#39;ve reviewed the procedures, let&#39;s look at some of the useful applications of co-immunoprecipitation and pull-down assays.
Co-immunoprecipitation can be useful in better understanding of enzymes&#39; mechanism of action. Myxovirus-, or Mx-, resistance proteins inhibit a wide range of viruses, including influenza A, for which the mechanism is poorly understood. Co-immunoprecipitation was used to study the interaction between mouse Mx1 protein and influenza nucleoprotein.
Pull-down assays have proven useful in studying the effects of second messengers, which are proteins that communicate a signal from the cellular environment. They are a component of a signaling pathway where multiple proteins interact in response to environmental cues. Calcium ions act as secondary messengers by binding to calmodulin, which, in turn, binds to a wide variety of proteins, mediating many types of biological responses. Without the calcium, the protein can&#39;t bind to the calmodulin. A pull-down assay was conducted to test the ability of proteins to bind to calmodulin in the presence or absence of calcium ions.
Co-immunoprecipitation and pull-down assays are generally used for analyzing stable or strong protein interactions, but not transient ones. A recent development in pull-down assays, the HaloTag, has simplified the study of transient protein interactions; HaloTag is a genetically-encoded protein fusion tag, fused to the protein of interest, capable of chemically reacting with a haloalkane solid support. If functional analysis is desired, the protein complex, minus the tag, in its entirety could then be isolated by incubating with tobacco etch virus protease.
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You&#39;ve just watched JoVE&#39;s video on co-immunoprecipitation and pull-down assays. This video described the principles of the two methods, the general laboratory procedures, and some of their applications.
Thanks for watching!
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Co-immunoprecipitation (CoIP) and pull-down assays are closely related methods to identify stable protein-protein interactions. These methods are related ...