Name: Purification of the Sarco-Endoplasmic Reticulum Ca2+-ATPase from Rabbit Muscle (Video) | JoVE Journal
Uploaded: 2025-03-21T14:00:00.000+00:00
Duration: 8 min 37 s
Description: Learn SERCA purification from rabbit muscle using trehalose stabilization for high-purity protein extraction. This method enables structural and functional studies of calcium transport mechanisms.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

The author acknowledges the help of Edmundo Mata-Morales in editing the video, VM, Valent&#237;n de la Cruz-Torres in purifying the SRVs, Miguel A. Rivera-Moran in SERCA purification and analysis, and Juan C. Gonzalez-Castro, Franco E. Juarez, Alejandra&#160;Nevarez, Nicolas&#160;Rocha-Vizuet, and Jocelin I. Ram&#237;rez-Alonso in video production. No funds, grants, or other support were received to conduct this study.

All animal procedures were performed in accordance with international and local (NORMA Oficial Mexicana NOM-062-ZOO-1999) guidelines for the handling of animals in experimental laboratories51,67,68. Muscle tissue was obtained from wild-type Oryctolagus cuniculus from a local animal handling unit (INE/CITES/DGVS-ZOO-E0055-SLP-98)46. A veterinarian with expertise in laboratory animal management performed the initial muscle dissection and processing. The details of the reagents and the equipment used in this study are listed in the Table of Materials.
1. Isolation of sarcoplasmic reticulum from fast-twitch rabbit muscle (Timing, 7 - 9 h)
NOTE: For details on the procedure, refer to Champeil et al.69.
<ol>
	<li>Obtain fast-twitch muscle tissue (~70 g from both hind legs per animal) from wild-type Oryctolagus cuniculus. 
	NOTE: Prior visual identification of the muscles to be dissected is required70,71.</li>
	<li>Macerate the fast-twitch muscle in a blender by suspending it in three volumes of 100 mM of KCl, with 1 min on and 1 min in ice-cold rest, repeat three times72.</li>
	<li>Remove the tissue debris by centrifugation (1500 × g) at 4 °C for 5 min; a second centrifugation (4200 × g) is performed if necessary.</li>
	<li>Collect the supernatant and homogenize it using a tissue grinder (10 strokes at low velocity, i.e., 500-1,500 rpm)72,73. Centrifuge the homogenate (10,000 × g) at 4 °C for 15 min. 
	NOTE: Care must be taken when homogenizing, as the use of a relatively high speed can result in damage to the system and the operator.</li>
	<li>Centrifuge the supernatant (containing the sarcoplasmic reticulum) (33,000 × g) at 4 °C for 120 min.</li>
	<li>Suspend the pellets in 40 mL of 0.5 M of sucrose and centrifuge (12,000 × g) at 4 °C for 15 min. Dilute the supernatant (0.6 M of KCl and 0.15 M of sucrose) and centrifuge the suspension (34,000 × g) at 4 °C for 165 min.</li>
	<li>Suspend the pellet containing the sarcoplasmic reticulum vesicles (SRVs) sheets in 0.3 M of sucrose, 0.1 M of KCl, and 5 mM of Tris-HCl, pH 7.0. Determine the protein concentration by using the Lowry assay74 and human serum albumin as the protein standard.</li>
	<li>Aliquot the SRVs (30-32 mg/mL) in 1 mL volume. Store the SRVs samples at -72 °C for further processing. 
	NOTE: Protein yield is 100-110 mg SRVs per animal.</li>
</ol>
2. SERCA purification (Timing, 18 - 20 h)
NOTE: For details on the procedure, refer to Rivera-Morán et al.46.
<ol>
	<li>Dilute the SRVs suspension to 2 mg protein/mL using ice-cold 75 mM of Tris-HCl, pH 7.2, 0.6 M of KCl, 6 mM of EDTA, 1 mM of EGTA, and 0.1% (w/v) deoxycholate (DOC). Incubate the SRVs suspension on ice for 10 min and gentle agitation. Centrifuge the SRVs suspension (100,000 × g) at 4 °C for 60 min. 
	NOTE: DOC should be added drop by drop.</li>
	<li>Suspend the pellets in &lt;3 mL of 25 mM of Tris-HCl, pH 7.5, 0.3 M of KCl, 45% glycerol (v/v), and 2 mM EDTA. Homogenize the suspension and adjust to a final volume of 10 mL. Determine protein concentration and then, adjust it to 6.5 mg/mL. Add azolectin at 5 mg/mL and 0.85 (w/w) Zwittergent 3-14. 
	NOTE: Azolectin and Zwittergent 3-14 should be added drop by drop.</li>
	<li>Homogenize the suspension and centrifuge (100,000 × g) at 4 °C for 60 min. Collect the supernatant and dilute 1:2 with 2 mM of EGTA (pH 7.2).</li>
	<li>Pour the suspension gently onto a discontinuous trehalose concentration gradient (45, 40, 35, and 30% w/v) in 10 mM of Tris-HCl, pH 7.0, 1 mM of EDTA, 0.1% deoxycholate and 1 mg/mL azolectin. Centrifuge (100,000 × g) at 4 °C for 14 h. Collect the transparent, slightly yellowish pellet formed at the bottom of the tubes. 
	NOTE: Both the formation of the trehalose gradient and the addition of the sample suspension must be performed using appropriate techniques. Overnight centrifugation is recommended.</li>
	<li>Suspend gently the pellet in a small volume (&lt;1.5 mL) of 25 mM of Tris-HCl, pH 7.5, 0.3 of M KCl, 45% glycerol, and 2 mM of EDTA. Determine protein concentration. Adjust the protein concentration by dilution to 2 mg/mL. Take aliquots (~50 µL) of the suspension and store the purified SERCA at −72 °C until its use.</li>
	<li>Perform SDS-PAGE of the purified SERCA72, and of protein samples taken at different steps of purification. Stain the gel with Coomassie Blue72. 
	NOTE: The use of 0.6 M trehalose instead of glycerol in the suspension buffer increases the stability of the enzyme during storage and freeze-thaw cycles28,29.</li>
</ol>
3. Assay for ATPase activity (Timing, 1 - 2 h)
<ol>
	<li>Enzyme-coupled assay: ATPase-Piruvate kinase-lactate dehydrogenase
	<ol>
		<li>Prepare the reaction mixture (1 mL): 50 mM MOPS (pH 7.0), 1 mM EGTA, 80 mM KCl, 5 mM MgCl2, 3 mM CaCl2, 5 mM phosphoenolpyruvate, 250 µM NADH. Include variable ATP concentrations (0.001 to 0.25 mM).</li>
		<li>Homogenize carefully the reaction assay by vortexing. Incubate the reaction assay at 37 ◦C for 10 min. Add 0.9 U L-lactate dehydrogenase (LDH) and 1.5 U pyruvate kinase (PK).</li>
		<li>Initiate the ATPase reaction by the addition of 10 µg of SERCA.</li>
		<li>Determine the formation of NADH. Follow the change in absorbance intensity every second for 600 s at a wavelength (λ) of 340 nm over time using a spectrophotometer with a thermostatted cell holder. 
		NOTE: The time interval for the absorbance measurement can vary, but try to record as many data points as possible to draw the initial straight line of the ATPase reaction.</li>
	</ol>
	</li>
	<li>Determine the rate of ATP hydrolysis 
	NOTE: For details on the procedure, refer to Rivera-Morán et al.46.
	<ol>
		<li>Calculate the slope value (Δabs/min) of the linear portion in each curve formed. Use the molar extinction coefficient (ε) of NADH (λ = 6,220 M−1·cm−1) to determine the ATPase activity (µmoles ATP hydrolyzed/min.mg prot.)</li>
	</ol>
	</li>
	<li>Determine the kinetics parameters Km and Vmax
	<ol>
		<li>Plot the velocity data versus ATP concentration using data analysis and graphing software.</li>
		<li>Fit the data by non-linear regression to the Michaelis-Menten Equation (Eq. 1)46: 
		<img alt="Michaelis-Menten equation: v=Vmax·S/(Km+S), enzyme kinetics formula, biochemical analysis." src="/files/ftp_upload/67748/67748eq01.jpg" style="margin: auto;" data-altupdatedat="1788304762000" data-alt="Equation 1"> 
		where v is the velocity, Vmax is the maximum velocity, [S] is the ATP concentration, and Km is the Michaelis-Menten constant.</li>
		<li>Determine Km and Vmax by iteration. 
		NOTE: The inclusion of DOC may increase the ATPase rate response; testing different concentrations of DOC is recommended to find the optimum.</li>
	</ol>
	</li>
</ol>
4. Labeling assay of SERCA with Fluorescein Isothiocyanate (FITC ) (Timing, 30 - 45 min, only for FITC labeling)
NOTE: For details on the procedure, refer to54,75,76.
<ol>
	<li>Suspend SERCA (20 µg) in 50 µL (final volume) of labeling buffer (100 mM of KCl, 5 mM MgCl2, and 30 mM of Tris-HCl, pH 8.9) containing 1 mM of FITC. Mix by vortexing.</li>
	<li>Incubate the samples at different times (15 min, 10 min, 7 min, 5 min, and 2 min) in the dark and at room temperature.</li>
	<li>Stop the labeling reaction by adding 1 volume of ice-cold stopping buffer (480 mM of sucrose and 48 mM of MOPS, pH 7.0), containing ATP (5 mM). Incubate on ice for 5 min in the dark.</li>
	<li>Subject the FITC-labeled SERCA to SDS-PAGE72. Expose the clear gels to UV light (λ = 302 nm). Photo document the result.</li>
	<li>Stain the gel with Coomassie blue. Photo document the result. 
	NOTE: Performing FITC labeling in a dark room may improve the quality of the fluorescence signal.</li>
</ol>
5. Circular dichroism (CD) spectra (Timing, 30 min)
NOTE: For details on the procedure, refer to11,77.
<ol>
	<li>Suspend SERCA (3 µM) in 400 µL of 10 mM phosphate buffer (pH 7.0) at 25 °C.</li>
	<li>Load the sample in a 0.1 cm path-length cell. Set far-UV spectra λ range between 190-260 nm. Set the internal resolution and the bandwidth to 1 nm.</li>
	<li>Record the CD signal at 50 nm/min at 25 °C.</li>
</ol>

The author declares that he has no competing financial interests.

Most molecules and ions cannot freely cross the cell membranes, e.g., proton (H+) requires a membrane transporter in the plasma membrane of a variety of organisms and organelles such as mitochondria83,84. Cell membranes are selective, and the molecules and ions that cross the cell membranes are diverse, so several types of membrane proteins can be found in the cell, such as (a) ABC transporters, (b) ion channels, (c) membrane-bound ATPases, (d) SLC tra...

Membrane proteins/enzymes are essential biological components of cells as they play critical roles in various processes1,2,3. Some of the functions may include transport of ions and molecules in and out of the cell/internal compartments (either active/passive), cell-cell recognition, intercellular binding, anchorage/attachment, and sensing of the external environment through integration with the signal transduction machinery under normal and harsh physical and chemical conditions (high salt, low water, high temperature, drug resistance, etc.)3&#160;Therefore, the determination of the three-dimensional (3D) structure of membrane proteins and/or enzymes has become of great importance for both basic and applied research4,7,6. Importantly, membrane proteins/enzymes have been extensively used as targets for drug discovery (whether natural or by design)7,8,9. That is, membrane proteins have an inherent importance in health9,10,11.
The hydrophobic character of membrane proteins/enzymes is the most technically challenging physicochemical property for the experimental laboratory12,13,14, even more so when working with oligomeric and/or highly labile integral membrane proteins15,16. The isolation of appropriate amounts of the membrane protein/enzyme with the highest possible quality for functional experimental assays and structural studies is highly desirable17. Membrane proteins, being inherently hydrophobic, are very difficult to purify, and the choice of detergent is usually one of the most important issues to be considered17,18,19,20. In this regard, the laboratory methods used to isolate membrane proteins typically cause some degree of damage to the 3D structural arrangement of the protein21. Some of these methods include the use of (a) sonication (high frequency/energy ultrasonic waves), (b) protein-solubilizing detergents (either harsh, medium, or gentle)22, (c) relatively high pressures in column chromatography and high-speed ultracentrifugation23, (d) precipitating molecules, (e) digestive enzymes, and others21. All these processes can contribute to or be the main cause of protein destabilization during purification21. In this respect, some protocols seem to work relatively well for a given membrane protein. However, optimization is always welcome when using new modern assays or methods that require a higher quality of membrane protein preparation to obtain satisfactory results24. Optimization steps may include, but are not limited to, improving construct design, finding optimal conditions for membrane protein expression, establishing better handling conditions (i.e., pH, temperature, etc.), finding the best compatible detergent, adjusting purification steps such as sonication time, centrifugation speed, reformulating buffer solutions by adding stabilizing agents, etc.25,26,27. Therefore, any change in the purification methodology that leads to an increase in the quality (purity) and activity of the purified membrane protein/enzyme is important.
In the P-type ATPase family, the yeast plasma membrane H+-ATPase appears to be one of the most labile members28,29. The H+-ATPase loses its ATP hydrolyzing activity upon dehydration (freeze-drying), heat shock, etc.28,29. The use of trehalose as a protein stabilizer was tested in the isolation of the plasma membrane H+-ATPase from the yeast K. lactis30,31; the H+-ATPase preparation obtained was of high purity and catalytically active. Importantly, this allowed the oligomeric state of the enzyme to be resolved by biochemical methods, revealing it to be a hexamer and later confirmed by cryo-electron microscopy31,32,33,34,35. Therefore, it seems likely that the delicate three-dimensional (3D) arrangement of membrane proteins can be lost under relatively harsh conditions during purification36. Biphasic inactivation kinetic is observed for H+-ATPase during heat-mediated inactivation29. In yeast cells, as in many other organisms, the disaccharide trehalose accumulates at high concentrations under environmental stress conditions37,38,39. Trehalose maintains membrane integrity and transport function by stabilizing proteins (both membrane-embedded and cytosolic) and cell membranes40,41. The stabilizing mechanism of trehalose has been extensively studied by several groups and by our laboratory42,43,44,45. Experiments with the H+-ATPase and other enzymes have shown that trehalose is the most effective protein stabilizer among mono- and disaccharides28,29. This led to its inclusion in the H+-ATPase purification protocol30. Recently, trehalose has also been used in the purification of the sarcoplasmic reticulum Ca2+-ATPase (SERCA) from rabbit fast-twitch muscle with good results in protein purity and activity46. Therefore, trehalose seems to be a good and appropriate additive for the purification of P-type ATPases and probably other membrane and cytosolic proteins.
For P-ATPases, the existence of structural cytoplasmic domains is an experimental advantage, especially for substrate/ligand interaction studies47; ATP binding has been studied in highly pure recombinant N-domains47,48,49, thus eliminating the technical considerations for purification of whole membrane enzymes47,48,50, among others51. Unfortunately, some functional (catalytic/energy conversion) and structural (subunit arrangement and interaction with other proteins) studies still require the whole P-ATPase52,53. In this regard, the purification of SERCA has been achieved by several research groups54,55,56,57,58. However, improvements can still be implemented46, for example, increasing the intactness of the purified ATPase59, avoiding protein denaturation/disruption of protein complexes25, increase solubilization without denaturation of the membrane protein (i.e., avoiding formation of macromolecular aggregates)60, lead to better compatibility with assays and other downstream analytical methods61&#160;Furthermore, as new experimental strategies, additives, enzyme inhibitors, etc. appear in the scientific literature61,62,63,64,65,66, they&#160;sometimes need to be tested with the whole P-ATPase. This work describes the protocol for the purification of SERCA and the use of trehalose as an additive to stabilize the protein structure and the ATPase activity; i.e., in addition to increasing the enzyme (structural) quality, trehalose helps to prevent the loss of enzyme structure and activity during enzyme isolation and storage, which helps to save biological material and thus reduce the number of enzyme purifications.

<table><tbody><tr><td>ATP</td><td>Sigma-Aldrich Corp</td><td>A2383</td><td></td></tr><tr><td>Azolectin from soybean</td><td>Sigma-Aldrich Corp</td><td>44924</td><td></td></tr><tr><td>Benchtop UV transilluminator&amp;nbsp;</td><td>Cole-Parmer</td><td>EW-97623-08</td><td>Dual intensity High setting is ideal for analytical documentation. Low setting reduces photonicking or photobleaching of gel samples while doing preparative work.</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; &amp;bull; 2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich Corp</td><td>223506</td><td></td></tr><tr><td>Coolpix B500 camera&amp;nbsp;</td><td>Nikon Corp</td><td>S210</td><td></td></tr><tr><td>Coomassie brilliant blue G-250</td><td>Bio-Rad</td><td>1610406</td><td></td></tr><tr><td>Dodecyl maltoside</td><td>Sigma-Aldrich Corp</td><td>D4641</td><td></td></tr><tr><td>Ethylene glycol-bis(2-aminoethylether)-&lt;em&gt;N,N,N&amp;prime;,N&amp;prime;&lt;/em&gt;-tetraacetic acid</td><td>Sigma-Aldrich Corp</td><td>E4378</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid tetrasodium salt dihydrate&amp;nbsp;</td><td>Sigma-Aldrich Corp</td><td>E6511</td><td></td></tr><tr><td>Fluorescein isothiocyanate</td><td>Sigma-Aldrich Corp</td><td>F3651</td><td></td></tr><tr><td>KCl</td><td>JT Baker</td><td>7447-40-7</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; &amp;bull; 6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich Corp</td><td>M9272</td><td></td></tr><tr><td>MOPS</td><td>Sigma-Aldrich Corp</td><td>M1254</td><td></td></tr><tr><td>NADH</td><td>Chem-Impex International Inc</td><td>230</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-tetradecyl-&lt;em&gt;N,N&lt;/em&gt;- dimethyl-3-ammonium-1-propanesulfonate</td><td>Sigma-Aldrich Corp</td><td>D0431</td><td></td></tr><tr><td>Phosphoenolpyruvate (PEP)</td><td>Chem-Impex International Inc</td><td>9711</td><td></td></tr><tr><td>Rabbit muscle lactate dehydrogenase</td><td>Roche</td><td>10003557103</td><td></td></tr><tr><td>Rabbit muscle pyruvate kinase</td><td>Sigma-Aldrich Corp</td><td>P1506</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma-Aldrich Corp</td><td>D6750</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Bio-Rad</td><td>1610302</td><td></td></tr><tr><td>Spectropolarimeter&amp;nbsp;</td><td>Jasco Corp.</td><td>Jasco J1500&amp;nbsp;</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich Corp</td><td>84100</td><td></td></tr><tr><td>Trehalose</td><td>Sigma-Aldrich Corp</td><td>T0167</td><td>Dihydrate</td></tr><tr><td>Tris(hydroxymethyl)aminomethane hydrochloride</td><td>Sigma-Aldrich Corp</td><td>857645</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman</td><td>Optima XPN</td><td></td></tr><tr><td>UV/VIS spectrophotometer&amp;nbsp;</td><td>Agilent Technologies</td><td>8453</td><td>The Agilent 8453 UV-Vis Spectrophotometer uses a photodiode array for simultaneous measurement of the complete ultra-violet to visible light spectrum</td></tr><tr><td>WiseStir HS- 30E</td><td>Daihan Scientific Co.</td><td>DH.WOS01010</td><td>Ideal for all disperging and homogenizing applications, designed for tissue grinders.</td></tr></tbody></table>

purification of the sarco-endoplasmic reticulum ca2+-atpase from rabbit muscle

Purification of the Sarco-Endoplasmic Reticulum Ca2+-ATPase from Rabbit Muscle

Some P-type ATPases, such as sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), are inherently labile membrane proteins that require specific physicochemical conditions during purification to obtain them with high purity and structural quality and in a catalytically active form. The disaccharide trehalose is a compatible solute that is synthesized and accumulated in high concentrations in the yeast cytoplasm to stabilize the membranes and proteins. The use of trehalose as an additive in the protocol for the purification of plasma membrane H+-ATPase results in a high-quality preparation, the hexameric structure of which is shown by biochemical analytical methods. Trehalose can, therefore, be used as a stabilizing additive for the purification of membrane proteins (P-ATPases). This protocol describes the modification of the classical protocol for SERCA purification by subjecting SERCA to centrifugation on a trehalose concentration gradient. The inclusion of this carbohydrate led to the purification of SERCA in a catalytically active form with high purity and, importantly, in a stable form. Partial biochemical characterization of the purified SERCA (SDS-PAGE, enzyme kinetics, FITC labeling, circular dichroism spectroscopy) showed that the enzyme is suitable for functional and structural studies. The use of trehalose in the purification protocol of P-type ATPases and other labile membrane (and cytosolic) proteins is suggested.

SDS-PAGE of SERCA at different stages of purification (Figure 1). The Coomassie blue-stained gel shows the enrichment of the SERCA protein band (apparent molecular weight &gt;100 kDa) as the purification protocol progresses. The protein band corresponding to SERCA shows a purity of &gt;90% after centrifugation on a trehalose concentration gradient. Plot of ATP hydrolysis rate versus ATP concentration (Figure 2A). Michaelis-Menten ATPase kinetic (hyperbo...

This protocol describes an improved SERCA purification method, which includes the disaccharide trehalose in the final centrifugation step. This carbohydrate stabilizes proteins under harsh conditions. The purified SERCA was catalytically active and displayed high purity, making it suitable for structural and functional studies.

Some P-type ATPases, such as sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), are inherently labile membrane proteins that require specific ...

purification-sarco-endoplasmic-reticulum-ca2-atpase-from-rabbit

Research

JoVE Journal

Biochemistry

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies.  A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store.   This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Here, we describe a protocol to evaluate sarcoplasmic reticulum Ca2+ reserve and diastolic calcium removal function in cardiomyocytes by a calcium imaging system.

Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

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.

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

This protocol describes the purification of F1-ATPase from the cultured insect stage of Trypanosoma brucei. The procedure yields a highly pure, homogeneous, and active complex suitable for structural and enzymatic studies.

Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

Mitochondria are key metabolic organelles that exhibit a high level of phenotypic plasticity in skeletal muscle. The import of proteins from the cytosol is a critical pathway for organelle biogenesis, essential for the expansion of the reticulum and the maintenance of mitochondrial function.  Therefore, protein import serves as a barometer of cellular health.

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

Functional site-directed fluorometry is a method to study protein domain motions in real time. Modification of this technique for its application in native cells now allows the detection and tracking of single voltage-sensor motions from voltage-gated Ca2+ channels in murine isolated skeletal muscle fibers.

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability

This protocol details the procedures for recombinantly producing the human myosin-7a holoenzyme using the MultiBac Baculovirus system and for studying its motility using a tailored in vitro filament gliding assay.

MultiBac System-Based Purification and Biophysical Characterization of Human Myosin-7a

Learn SERCA purification from rabbit muscle using trehalose stabilization for high-purity protein extraction. This method enables structural and functional studies of calcium transport mechanisms.

Purification of the Sarco-Endoplasmic Reticulum Ca²⁺-ATPase from Rabbit Muscle

Explore More Videos