Name: enrichment of mammalian tissues and xenopus oocytes with cholesterol
Uploaded: 2020-03-25T13:00:00
Description: Watch this Scientific Journal Video about Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol at JoVE.com

This work was supported by a Scientist Development Grant (11SDG5190025) from the American Heart Association (to A.R.-D.), and by National Institute of Health R01 grants AA-023764 (to A.N.B.), and HL-104631 and R37 AA-11560 (to A.M.D).

All experimental procedures with animals were performed at the University of Tennessee Health Science Center (UTHSC). The care of animals and experimental protocols were reviewed and approved by the Animal Care and Use Committee of the UTHSC, which is an institution accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.
1. Enrichment of tissues and cells using methyl-&#946;-cyclodextrin saturated with cholesterol
NOTE: ...

Methods to enrich mammalian tissues and cells and Xenopus oocytes with cholesterol constitute a powerful tool for investigating the effect of elevated cholesterol levels on individual molecular species, on complex macromolecular systems (e.g., proteins), and on cellular and organ function. In this paper, we have described two complementary approaches that facilitate such studies. First, we described how to enrich tissues and cells with cholesterol using M&#946;CD saturated with cholesterol. We demonstrated that ...

Cholesterol, a major cellular lipid, plays numerous critical functional and structural roles1,2,3,4,5,6,7,8,9. From regulating the physical properties of the plasma membrane to ensuring cell viability, growth, proliferation, and serving as a signaling and precursor molecule in a plethora of biochemical pathways, cholesterol is an imperative component necessary for normal cell and organ function. As a result, cholesterol deficiency results in severe physical malformations and a variety of disorders. On the other hand, even a small increase in cholesterol above physiological levels (2-3x) is cytotoxic1,2,10 and has been associated with the development of disorders, including cardiovascular11,12,13 and neurodegenerative diseases14,15,16,17. Thus, to interrogate the critical functions of cholesterol and to determine the effect of changes in cholesterol levels, different approaches that alter the content of cholesterol in tissues, cells, and Xenopus oocytes have been developed.
Alteration of cholesterol levels in mammalian tissues and cells 
Several approaches can be harnessed to decrease the levels of cholesterol in tissues and cells18. One approach involves their exposure to statins dissolved in lipoprotein-deficient serum to inhibit HMG-CoA reductase, which controls the rate of cholesterol synthesis19,20. However, these cholesterol lowering drugs also inhibit the formation of non-sterol products along the mevalonate pathway. Therefore, a small amount of mevalonate is added to allow the formation of these products21 and enhance the specificity of this approach. Another approach for decreasing cholesterol levels involves the use of &#946;-cyclodextrins. These glucopyranose monomers possess an internal hydrophobic cavity with a diameter that matches the size of sterols22, which facilitates the extraction of cholesterol from cells, thereby depleting them from their native cholesterol content23. An example is 2-hydroxypropyl-&#946;-cyclodextrin (HP&#946;CD), a preclinical drug currently being tested for treatment of the Niemann-Pick type C disease, a genetically inherited fatal metabolic disorder characterized by lysosomal cholesterol storage24. The level of cholesterol depletion depends on the specific derivative used. For example, HP&#946;CD extracts cholesterol with a lower capacity than the methylated derivative, methyl-&#946;-cyclodextrin (M&#946;CD)24,25,26,27,28,29,30. Notably, however, &#946;-cyclodextrins can also extract other hydrophobic molecules in addition to cholesterol, which may then result in nonspecific effects31. In contrast to depletion, cells and tissues can be specifically enriched with cholesterol through treatment with &#946;-cyclodextrin that has been presaturated with cholesterol23. This approach can also be used as a control for the specificity of &#946;-cyclodextrins used for cholesterol depletion31. Depletion of cholesterol from tissues and cells is straightforward and can be achieved by exposing the cells for 30-60 min to 5 mM M&#946;CD dissolved in the medium used for storing the cells. This approach can result in a 50% decrease in cholesterol content (e.g., in hippocampal neurons32, rat cerebral arteries33). On the other hand, preparing the &#946;-cyclodextrin-cholesterol complex for cholesterol enrichment of tissue and cells is more complex, and will be described in the protocol section.
An alternative approach to enriching tissues and cells using &#946;-cyclodextrin saturated with cholesterol involves the use of LDL, which relies on LDL receptors expressed in the tissues/cells18. While this approach offers the advantage of using the natural cholesterol homeostasis machinery of the cell, it has several limitations. First, tissues and cells that do not express the LDL receptor cannot be enriched using this approach. Second, LDL particles contain other lipids in addition to cholesterol. Specifically, LDL is comprised of the protein ApoB100 (25%) and the following lipids (75%): ~6-8% cholesterol, ~45-50% cholesteryl ester, ~18-24% phospholipids, and ~4-8% triacylglycerols34. Thus, delivery of cholesterol via LDL particles is nonspecific. Third, the percentage of increase in cholesterol content by LDL in tissues and cells that express the LDL receptor may be significantly lower than the increase observed using cyclodextrin saturated with cholesterol. For example, in a previous study, enrichment of rodent cerebral arteries with cholesterol via LDL resulted in only a 10-15% increase in cholesterol levels35. In contrast, enrichment of these arteries with cyclodextrin saturated with cholesterol as described in the protocol section resulted in &#62;50% increase in the cholesterol content (See Representative Results section, Figure 1).
Alteration of cholesterol levels in Xenopus oocytes 
Xenopus oocytes constitute a heterologous expression system commonly used for studying cell and protein function. Earlier studies have shown that the cholesterol to phospholipid molar ratio in Xenopus oocytes is 0.5 &#177; 0.136. Due to this intrinsic high level of cholesterol, increasing the content of cholesterol in this system is challenging, yet can be achieved using dispersions made from membrane phospholipids and cholesterol. The phospholipids that we have chosen for this purpose are similar to those used for forming artificial planar lipid bilayers and include L-&#945;-phosphatidylethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS), as described in the protocol section. This approach can result in &#62;50% increase in cholesterol content (See Representative Results section, Figure 2).
An alternative approach to enriching Xenopus oocytes with phospholipid-based dispersions involves the use of cyclodextrin saturated with cholesterol, which is similar to the way tissues and cells are enriched. However, we have found this approach to be of low reproducibility and efficiency, with an average of ~25% increase in cholesterol content. This is possibly due to the different loading capacity of these two approaches (See Representative Results section, Figure 3). In contrast, it has been shown that using cyclodextrin to deplete cholesterol from Xenopus oocytes can result in a ~40% decrease in cholesterol content36.
Here, we focus on cholesterol enrichment of mammalian tissues and cells through the application of cyclodextrin saturated with cholesterol, and of Xenopus oocytes using liposomes. Both approaches can be harnessed to delineate the effect of increased levels of cholesterol on protein function. The mechanisms of cholesterol modulation of protein function may involve direct interactions8 and/or indirect effects9. When cholesterol affects protein function via direct interactions, the effect of an increase in cholesterol levels on protein activity is likely independent of the cell type, expression system, or enrichment approach. For example, we utilized these two approaches to determine the effect of cholesterol on G-protein gated inwardly rectifying potassium (GIRK) channels expressed in atrial myocytes37, hippocampal neurons32,38, HEK29339 cells, and Xenopus oocytes32,37. The results obtained in these studies were consistent: in all three types of mammalian cells and in amphibian oocytes cholesterol upregulated GIRK channel function (see Representative Results section, Figure 4, for hippocampal neurons and the corresponding experiments in Xenopus oocytes). Furthermore, the observations made in these studies were also consistent with the results of studies carried out in atrial myocytes37,40 and hippocampal neurons32,38 freshly isolated from animals subjected to a high cholesterol diet40. Notably, cholesterol enrichment of hippocampal neurons using M&#946;CD reversed the effect of atorvastatin therapy used for addressing the impact of the high cholesterol diet both on cholesterol levels and GIRK function38. In other studies, we investigated the effect of mutations on cholesterol sensitivity of the inwardly rectifying potassium channel Kir2.1 using both Xenopus oocytes and HEK293 cells41. Again, the effect of the mutations on the sensitivity of the channel was similar in the two systems.
The applications of both enrichment methods for determining the impact of elevated cholesterol levels on molecular, cellular, and organ function are numerous. In particular, the use of cyclodextrin-cholesterol complexes to enrich cells and tissues is very common largely due to its specificity. Recent examples of this approach include the determination of the impact of cholesterol on HERG channel activation and underlying mechanisms42, the discovery that cholesterol activates the G protein coupled receptor Smoothened to promote Hedgehog signaling43, and the identification of the role of cholesterol in stem cell biomechanics and adipogenesis through membrane-associated linker proteins44. In our own work, we utilized mammalian tissue enrichment with the M&#946;CD:cholesterol complex to study the effect of cholesterol enrichment on basic function and the pharmacological profile of calcium- and voltage-gated channels of large conductance (BK, MaxiK) in vascular smooth muscle35,45,46. In other studies, we used the phospholipid-based dispersion approach for enriching Xenopus oocytes with cholesterol to determine the roles of different regions in Kir2.1 and GIRK channels in cholesterol sensitivity41,47,48,49, as well as to determine putative cholesterol binding sites in these channels32,50,51.

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			<td>Amplex Red Cholesterol Assay Kit</td>
			<td>Invitrogen</td>
			<td>A12216</td>
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			<td>Thermo Scientific</td>
			<td>23225</td>
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			<td>Brain PE 25Mg in Chloroform</td>
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			<td>S6014</td>
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			<td>Fisher</td>
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			<td>Hamilton</td>
			<td>702</td>
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			<td>12 mL heavy duty conical centrifuge beaded rim tube</td>
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			<td>Support Stand</td>
			<td>Homescience Tools</td>
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			<td>B3D 1308</td>
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			<td>Glass beakers 40ml-1L</td>
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			<td>02-540</td>
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			<td>Fisher</td>
			<td>Model 100</td>
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			<td>Eppendorf microcentrifuge</td>
			<td>Eppendorf</td>
			<td>Model 5417R</td>
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			<td>Amber bottles</td>
			<td>Fisher</td>
			<td>03-251-420</td>
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			<td>Corning&#8482; Disposable Glass Pasteur Pipets</td>
			<td>FIsher</td>
			<td>13-678-4A</td>
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			<td>FIsher</td>
			<td>50-998-944</td>
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			<td>Isotemp&#8482; BOD Refrigerated Incubator</td>
			<td>FIsher</td>
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			<td>Oocytes</td>
			<td>Xenoocyte&#8482;</td>
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			<td>Methyl-&#946;-cyclodextrin</td>
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			<td>51221080</td>
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			<td>DMSO</td>
			<td>Fisher</td>
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			<td>Paraformaldehyde 4%</td>
			<td>Mallinckrodt</td>
			<td>2621</td>
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			<td>DI H2O</td>
			<td>University DI source</td>
			<td></td>
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		<tr>
			<td>ProLong Gold antifade reagnet</td>
			<td>Invitrogen</td>
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		<tr>
			<td>Microslides 75x25mm Frosted</td>
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			<td>G15978A</td>
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			<td>Forceps</td>
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			<td>Microscope Coverslip</td>
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			<td>Securline Lab Marker II</td>
			<td>Sigma</td>
			<td>Z648205-5EA</td>
			<td></td>
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			<td>BD 10mL Syringe</td>
			<td>Fisher</td>
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			<td>70% ETOH</td>
			<td>Pharmco</td>
			<td>211USP/NF</td>
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			<td>Timer</td>
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			<td>02-261-840</td>
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		<tr>
			<td>Steno book</td>
			<td>Staples</td>
			<td>163485</td>
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enrichment of mammalian tissues and xenopus oocytes with cholesterol

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of methods. Here, we describe two important approaches used for this purpose. First, we describe how to enrich tissues and cells with cholesterol using cyclodextrin saturated with cholesterol using cerebral arteries (tissues) and hippocampal neurons (cells) as examples. This approach can be used for any type of tissue, cells, or cell lines. An alternative approach for cholesterol enrichment involves the use of low-density lipoprotein (LDL). The advantage of this approach is that it uses part of the natural cholesterol homeostasis machinery of the cell. However, whereas the cyclodextrin approach can be applied to enrich any cell type of interest with cholesterol, the LDL approach is limited to cells that express LDL receptors (e.g., liver cells, bone marrow-derived cells such as blood leukocytes and tissue macrophages), and the level of enrichment depends on the concentration and the mobility of the LDL receptor. Furthermore, LDL particles include other lipids, so cholesterol delivery is nonspecific. Second, we describe how to enrich Xenopus oocytes with cholesterol using a phospholipid-based dispersion (i.e., liposomes) that includes cholesterol. Xenopus oocytes constitute a popular heterologous expression system used for studying cell and protein function. For both the cyclodextrin-based cholesterol enrichment approach of mammalian tissue (cerebral arteries) and for the phospholipid-based cholesterol enrichment approach of Xenopus oocytes, we demonstrate that cholesterol levels reach a maximum following 5 min of incubation. This level of cholesterol remains constant during extended periods of incubation (e.g., 60 min). Together, these data provide the basis for optimized temporal conditions for cholesterol enrichment of tissues, cells, and Xenopus oocytes for functional studies aimed at interrogating the impact of cholesterol enrichment.

The use of cyclodextrin saturated with cholesterol as a means for enriching tissues and cells with cholesterol is well established. Here, we first demonstrate the application of this widely used approach for enriching rat cerebral arteries with cholesterol using M&#946;CD saturated with cholesterol. Figure 1A shows an example of an imaged cerebral artery smooth muscle layer and demonstrates the concentration-dependent increase in filipin-asso...

Watch this Scientific Journal Video about Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol at JoVE.com

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Two methods of cholesterol enrichment are presented: the application of cyclodextrin saturated with cholesterol to enrich mammalian tissues and cells, and the use of cholesterol-enriched phospholipid-based dispersions (liposomes) to enrich Xenopus oocytes. These methods are instrumental for determining the impact of elevated cholesterol levels in molecular, cellular, and organ function.

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of ...

Elevated cholesterol is a major risk factor for cardiovascular and neurodegenerative disease. Our protocols provide valuable tools for studying both physiological and mechanistic consequences of hypercholesterolemia. These procedures can be performed using basic lab equipment, and are applicable to cells, tissues, and Xenopus oocytes.Cholesterol is a major component of cellular membranes throughout the body. These techniques can be utilized to study the impact of elevated levels of cholesterol in any cell type. Dissecting the arteries is the most difficult step.Carefully remove the artery from the brain, without stretching or damaging the vascular tissue. Lipids are very delicate, and working with them is more of an art than a science. Video demonstration provides a guide for learning how to handle lipids carefully.To prepare the cholesterol saturated methyl-beta-cyclodextrin solution, first add 064 grams of methyl-beta-cyclodextrin to 10 milliliters of PBS, stirring the solution with a stir bar, to ensure that the methyl-beta-cyclodextrin fully dissolves. Next, add 0024 grams of cholesterol powder to the flask, and stir the solution vigorously, using a spatula to break up as many cholesterol chunks as possible. When nearly all the cholesterol has been dissolved, seal the flask with at least two layers of paraffin film.And shake the flask at about 30 oscillations per minute in a 37 degree Celsius water bath overnight. After eight to 16 hours, cool the solution to room temperature, before filtering the mixture through a 22 micrometer polyethersulfone syringe filter into a class bottle. For mammalian cerebral artery enrichment, harvest the brain from a 250 to 300 gram Sprague Dawley rat into a beaker of PBS on ice, before transferring the brain into a waxed dissection bowl, under a dissecting microscope, containing just enough PBS to submerge the tissue.Secure the brain with two to three pins, making sure they do not penetrate blood vessels. And use sharp forceps and small surgical scissors to gently dissect the cerebral arteries and their branches that form the Circle of Willis at the base of the brain. Next, briefly rinse up to one centimeter long artery segments in PBS, before incubating the rinsed segments in enough cholesterol enriching solution to cover the tissues.To determine any alterations in cholesterol level, use a fresh bottle of filipin powder to prepare a 10 milligram per milliliter stock solution of the dye in dimethyl sulfoxide, and wash the cholesterol enriched tissues with three five minute washes in fresh PBS. After the last wash, fix the artery segments in 4%paraformaldehyde for 15 minutes on ice, protected from light, before permeabilizing the samples in 5%Triton in PBS for 10 minutes at room temperature. At the end of the incubation, wash the tissues with three five minute washes in PBS on a shaker, before adding the samples to a 25 microgram per milliliter final concentration of filipin dye solution, for one hour at room temperature, protected from light.At the end of the incubation, wash the artery pieces three times as demonstrated, followed by a brief rinse with distilled water. After the last wash, use a lab tissue to absorb any excess liquid, and mount the arteries onto a slide using an appropriate mounting medium. Carefully cover the arteries with a cover slip, taking care to avoid rolling or twisting of the tissue, and let the slide dry for 24 hours at room temperature, protected from light.When the mounting medium is dry, seal the cover slip edges with clear nail polish, and allow the polish to dry for 10 to 15 minutes. Then, image the tissue with an excitation of 340 to 380 nanometers, and an emission of 385 to 470 nanometers. To prepare the phospholipid based dispersion, combine 200 microliters of 10 milligram per milliliter chloroform dissolved lipid solution, L-alpha-phosphatidylethanolamine, 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, and cholesterol in a 12 milliliter glass tube.Evaporate the chloroform in the hood under a stream of nitrogen, before suspending the lipids in 800 microliters of buffered 150 millimolar potassium chloride and 10 millimolar Tris HEPES solution. Then seal the tube with paraffin film, and gently sonicate the tube contents at 80 kilohertz for 10 minutes, until a milky mixture is formed. For cholesterol enrichment of Xenopus oocytes, use sharp forceps to disrupt the ovarian sac from a female Xenopus laevis frog in multiple regions, and place the ovary chunks into a 60 millimeter plate with five milliliters of calcium free ND96 supplemented with 5 milligrams per milliliter of collagenase.Shake the tissue on an orbital shaker at 60 oscillations per minute for 15 minutes at room temperature. At the end of the incubation, use a transfer pipette with a wide tip to vigorously pipette the oocyte containing solution five to 10 times to isolate the individual oocytes, and quickly rinse the dark oocyte solution with fresh calcium free ND96 until the solution becomes transparent. Next, transfer 90 microliters of the cholesterol enriched phospholipid based dispersion into one well of a 96 well plate, and add up to six oocytes to the well with as little medium as possible.Place the 96 well plate onto a three dimensional platform rotator for five to 10 minutes. Then add a drop of ND96 to the well, and transfer the cholesterol enriched oocytes to a 35 millimeter plate containing ND96 for their immediate analysis. Here, an example of an imaged cerebral artery smooth muscle layer demonstrates the concentration dependent increase in filipin associated fluorescence obtained upon tissue enrichment with increasing concentrations of cholesterol.Notably three hours subsequent to the treatment with the methyl-beta-cyclodextrin cholesterol complex the cholesterol levels decreased by approximately 50%compared to their level immediately after the enrichment. While a one hour incubation time is commonly used to enrich the tissues and cells with cholesterol during this approach, five minutes of incubation is usually sufficient to achieve a statistically significant increase in cerebral arterial cholesterol content as determined by cholesterol oxidase based biochemical assay. While no significant change is observed in cholesterol levels in Xenopus oocytes enriched with control phospholipid based dispersions lacking cholesterol, cholesterol levels increase significantly after only five minutes of treatment with the phospholipid based dispersions that include cholesterol, and remain at this level when the incubation time is increased to 60 minutes.The effectiveness of the cyclodextrin based approach for enriching cells is also demonstrated in neurons freshly isolated from the CA1 region of the hippocampus. Indeed, incubation of the neurons in methyl-beta-cyclodextrin saturated with cholesterol for 60 minutes results in an over two times increase in cholesterol levels, as assessed by the filipin associated fluorescence. Overall, the two most important steps for these procedures are preparing the cholesterol enrichment mixture and ensuring the integrity of the arterial tissue.Following cholesterol enrichment, one can study the function of the proteins that are affected by hypercholesterolemia. The ability to enrich cells with cholesterol facilitated the study of elevated cholesterol levels in cardiomyocytes and neurons. The use of oocytes allowed us to investigate how cholesterol binds to ion channels.A lab coat and gloves she always be worn for this protocol. Chloroform and paraformaldehyde should be used under a fume hood and be discarded as hazardous waste.

Research

JoVE Journal

Biochemistry

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human ...

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...

Quantification of Coenzyme A in Cells and Tissues

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. ...