Name: a buoyancy-based method of determining fat levels in drosophila
Uploaded: 2016-11-01T11:15:00
Description: Watch this Scientific Journal Video about A Buoyancy-based Method of Determining Fat Levels in Drosophila at JoVE.com

K.E.H. was supported by the Training Grant in Molecular Biology NIH-T32-GM08730. This work was supported by NIH, NIDDK Grant 5K01DK095932 and AHA Award 12BGIA11930014 to T.R.

1. Samla Ägg från Flugor med genotyper av intresse OBS: Perfekt korsningar består av 150 jungfru flugor och minst 75 hanar. Lagersamlingar bör bestå av minst 200 flugor. <ol><li> Förbereda druv plattor. <ol><li> Lägga 37,5 g agar till 1,5 L destillerat vatten i en 4 liters kolv. </li><li> Autoklav vatten / agar mix för 50 minuter vid 121 ° C (250 F). </li><li> Medan vatten / agar mix är autoklavering, tillsätt 50 g sackaros till 500 ml druvsaft och rör om med en omrörare på en upphettad rör plat...

<ol>
	<li>Bier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila,+the+golden+bug,+emerges+as+a+tool+for+human+genetics.">Drosophila, the golden bug, emerges as a tool for human genetics.</a> Nat Rev Genet. 6, 9-23 (2005).</li><li>Liu, Z., Huang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+metabolism+in+Drosophila+development+and+disease+Using+Drosophila+System+to+Study+Lipid+Metabolism+Lipids+Function+in+Drosophila+Early+Development.">Lipid metabolism in Drosophila development and disease Using Drosophila System to Study Lipid Metabolism Lipids Function in Drosophila Early Development.</a> Acta Biochim Biophys Sin. 45 (1), 44-50 (2013).</li><li>Ruden, D. M., Lu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+conservation+of+metabolism+explains+howDrosophila+nutrigenomics+can+help+us+understand+human+nutrigenomics.">Evolutionary conservation of metabolism explains howDrosophila nutrigenomics can help us understand human nutrigenomics.</a> Genes Nutr. 1 (2), 75-84 (2006).</li><li>Wolf, M. J., Rockman, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila,+genetic+screens,+and+cardiac+function.">Drosophila, genetic screens, and cardiac function.</a> Circ. Res. 109, 794-806 (2011).</li><li>Trinh, I., Boulianne, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+obesity+and+its+associated+disorders+in+Drosophila.">Modeling obesity and its associated disorders in Drosophila.</a> Physiology (Bethesda). 28 (2), 117-124 (2013).</li><li>Azeez, O. I., Meintjes, R., Chamunorwa, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fat+body,+fat+pad+and+adipose+tissues+in+invertebrates+and+vertebrates:+the+nexus.">Fat body, fat pad and adipose tissues in invertebrates and vertebrates: the nexus.</a> Lipids Health Dis. 13, 71 (2014).</li><li>Dourlen, P., Sujkowski, A., Wessells, R., Mollereau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid+transport+proteins+in+disease:+New+insights+from+invertebrate+models.">Fatty acid transport proteins in disease: New insights from invertebrate models.</a> Prog. Lipid Res. 60, 30-40 (2015).</li><li>Hildebrandt, A., Bickmeyer, I., K&#252;hnlein, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+Drosophila+body+fat+quantification+by+a+coupled+colorimetric+assay.">Reliable Drosophila body fat quantification by a coupled colorimetric assay.</a> PLoS One. 6 (9), e23796 (2011).</li><li>Tennessen, J. M., Barry, W. E., Cox, J., Thummel, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+studying+metabolism+in+Drosophila.">Methods for studying metabolism in Drosophila.</a> Methods. 68, 105-115 (2014).</li><li>Al-Anzi, B., Zinn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+measurement+of+triglycerides+cannot+provide+an+accurate+measure+of+stored+fat+content+in+Drosophila.">Colorimetric measurement of triglycerides cannot provide an accurate measure of stored fat content in Drosophila.</a> PLoS One. 5 (8), e12353 (2010).</li><li>Thanh, N. T. K., Stevenson, G., Obatoml, D., Bach, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Lipids+in+Animal+Tissues+by+High-Performance+Thin-Layer+Chromatography+with+Densitometry.">Determination of Lipids in Animal Tissues by High-Performance Thin-Layer Chromatography with Densitometry.</a> J. Planar Chromatogr. 13, 375-381 (2000).</li><li>Borrull, A., Lopez-Martinez, G., Poblet, M., Cordero-Otero, R., Rozes, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+the+separation+and+quantification+of+neutral+lipid+species+using+GC-MS.">A simple method for the separation and quantification of neutral lipid species using GC-MS.</a> Eur. J. Lipid Sci. Technol. 117, 274-280 (2015).</li><li>Shui, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+one+step+analysis+of+cellular+lipidomes+using+liquid+chromatography+coupled+with+mass+spectrometry:+application+to+Saccharomyces+cerevisiae+and+Schizosaccharomyces+pombe+lipidomics.">Toward one step analysis of cellular lipidomes using liquid chromatography coupled with mass spectrometry: application to Saccharomyces cerevisiae and Schizosaccharomyces pombe lipidomics.</a> Mol. Biosyst. 6, 1008-1017 (2010).</li><li>Farvid, M. S., Ng, T. W. K., Chan, D. C., Barrett, P. H. R., Watts, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+adiponectin+and+resistin+with+adipose+tissue+compartments,+insulin+resistance+and+dyslipidaemia.">Association of adiponectin and resistin with adipose tissue compartments, insulin resistance and dyslipidaemia.</a> Diabetes. Obes. Metab. 7 (4), 406-413 (2005).</li><li>Urbanchek, M. G., Picken, E. B., Kalliainen, L. K., Kuzon, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+force+deficit+in+skeletal+muscles+of+old+rats+is+partially+explained+by+the+existence+of+denervated+muscle+fibers.">Specific force deficit in skeletal muscles of old rats is partially explained by the existence of denervated muscle fibers.</a> J. Gerontol. A. Biol. Sci. Med. Sci. 56A (5), B191-B197 (2001).</li><li>Mosher, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+between+Drosophila+Arc1+and+a+specific+population+of+brain+neurons+regulates+organismal+fat.">Coordination between Drosophila Arc1 and a specific population of brain neurons regulates organismal fat.</a> Dev. Biol. 405 (2), 280-290 (2015).</li><li>Reis, T., van Gilst, M. R., Hariharan, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+buoyancy-based+screen+of+drosophila+larvae+for+fat-+storage+mutants+reveals+a+role+for+Sir2+in+coupling+fat+Storage+to+Nutrient+Availability.">A buoyancy-based screen of drosophila larvae for fat- storage mutants reveals a role for Sir2 in coupling fat Storage to Nutrient Availability.</a> PLoS Genet. 6 (11), e1001206 (2010).</li><li>Teixeira, L., Rabouille, C., R&#248;rth, P., Ephrussi, A., Vanzo, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Perilipin/ADRP+homologue+Lsd2+regulates+lipid+metabolism.">Drosophila Perilipin/ADRP homologue Lsd2 regulates lipid metabolism.</a> Mech. Dev. 120 (9), 1071-1081 (2003).</li><li>Perez, C. L., Van Gilst, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+13C+Isotope+Labeling+Strategy+Reveals+the+Influence+of+Insulin+Signaling+on+Lipogenesis+in+C.+elegans.">A 13C Isotope Labeling Strategy Reveals the Influence of Insulin Signaling on Lipogenesis in C. elegans.</a> Cell Metab. 8, 266-274 (2008).</li></ol>

Det finns en mängd olika tekniker som har utvecklats för att mäta lipidnivåer 8-10. Emellertid kommer var och en av dessa metoder med några stora nackdelar som tas upp av flytkraften baserad analys som beskrivs ovan. För det första är denna analys extremt snabbt. Testa en full koncentration gradient tar inte mer än 30-60 minuter. Detta är en enorm förbättring jämfört med de flesta av de tekniker som för närvarande används. Till exempel, lipid kvantifiering av MS tar 7-9 ...

Drosophila melanogaster har använts i över ett sekel i studiet av genetik och andra grundläggande biologiska frågor. Under de senaste decennierna har det blivit tydligt att Drosophila är ett kraftfullt verktyg i studiet av många mänskliga sjukdomar. Som 70 - 80% av gener associerade med humana sjukdomar har en identifierad fly ortolog 1-4 ger Drosophila ett förenklat ännu översättnings system där för att studera komplexa sjukdomar. Metabolism i synnerhet har dragit nytta av sådan studie. Inte bara är genetiken av metabolism väl bevarade mellan flugor och människor, men de relevanta organen och celltyper är också mycket lik 2,5. Till exempel, det fett kroppen av flugan lagrar både triacylglycerider (TAG) och glykogen, funktioner som är analoga med de som utförs i däggdjurslever och vitt adiposvävnad 6. Med hjälp av Drosophila som en modell för mänsklig fetma har betydligt förbättrat vår förståelse av lipidmetabolism och genetik fetma 7. Larvstadiet i utvecklingen är särskilt användbar för att studera segregeringen av näringsämnen till lagring eller användning som den är tillägnad matning och lagring av energi som skall användas under pupariation. För närvarande finns det många olika kvantitativa metoder för att bestämma fettlagringsnivåer i Drosophila. Den mest använda metoden är den kopplade kolorimetrisk analys (CCA) 8,9. CCA var utvecklad för bestämning TAG-nivåer i humant serum och verkar på premissen att glycerol frigjord från ryggraden av triglycerider kommer att genomgå flera reaktioner, vilket slutligen resulterar i en redox-kopplad reaktion alstra en färgad produkt. Absorbans av specifika ljusvåglängder mäts sedan för att bestämma den initiala mängden av glycerol närvarande. Däremot kan glycerol även befrias från mono- och diacylglycerides förutom TAG och kan därför inte vara ett exakt mått på svakas kroppsfett 9. Dessutom kan ögat pigment krossade vuxna flugor störa vissa absorbansmätningar och komplicera resultat 9,10. Därför måste CCA åtföljas och validerats av tunnskiktskromatografi (TLC), som gör det möjligt att separera de flesta lipid familjer som kan kvantifieras genom densitometri 10,11. Dock kan vissa lipidklasser som steroler inte analyseras och måste kvantifieras på ett annat sätt 12. Masspektrometri (MS) är ett korrekt sätt att kvantifiera alla klasser av större cellulära lipider 12,13. Men lipidextraktion förfaranden som är nödvändiga för att analysera med MS är både tidskrävande (de flesta ta nästan en hel dag) och kostsamt. Här presenterar vi en alternativ metod för att snabbt och billigt bestämma organismfettnivåer i L3 larver av Drosophila melanogaster. Den metod som presenteras nedan utnyttjar skillnaden i densitet mellan fettvävnad och mager vävnad. däggdjurs fat vävnad har en densitet av ca 0,9 g / ml 14 medan skelettmuskulaturen som en densitet av 1,06 g / ml 15. Denna skillnad betyder att djur med högre butiker av fett kommer att ha lägre densitet än djur med lägre förråd av fett, vilket ger dem möjlighet att flyta bättre i en lösning av den fasta densiteten. Den här egenskapen möjliggör extremt snabb screening av ett stort antal djur och samtidigt vara både billig och icke-invasiv. Flytkraft baserad analys har använts både för att bekräfta fenotyper av att förändra kända regulatorer för fettnivåer samt att identifiera nya genetiska och neurologiska regulatorer av fetma 16,17.

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a buoyancy-based method of determining fat levels in drosophila

Drosophila melanogaster is a key experimental system in the study of fat regulation. Numerous techniques currently exist to measure levels of stored fat in Drosophila, but most are expensive and/or laborious and have clear limitations. Here, we present a method to quickly and cheaply determine organismal fat levels in L3 Drosophila larvae. The technique relies on the differences in density between fat and lean tissues and allows for rapid detection of fat and lean phenotypes. We have verified the accuracy of this method by comparison to body fat percentage as determined by neutral lipid extraction and gas chromatography coupled with mass spectrometry (GCMS). We furthermore outline detailed protocols for the collection and synchronization of larvae as well as relevant experimental recipes. The technique presented below overcomes the major shortcomings in the most widely used lipid quantitation methods and provides a powerful way to quickly and sensitively screen L3 larvae for fat regulation phenotypes while maintaining the integrity of the larvae. This assay has wide applications for the study of metabolism and fat regulation using Drosophila.

Figur 1 visar ett exempel på hur flytbaserad analys kan detektera både högre och lägre fettdepåer i genetiskt manipulerade larver. Figur 1A visar att excitation eller tysta en viss delmängd av nervceller (E347) i larv hjärnan framkallar lägre och högre fett butiker respektive. Samma genetiska bakgrund kontroll användes i båda fallen. Figur 1B ger ett exempel på hur denna fenotyp som erhållits genom analysen tätheten bekräf...

Watch this Scientific Journal Video about A Buoyancy-based Method of Determining Fat Levels in Drosophila at JoVE.com

A Buoyancy-based Method of Determining Fat Levels in Drosophila

Här presenterar vi en metod för att mäta organismfettnivåer i tredje stadiet (L3) larvstadiet av Drosophila melanogaster. Denna metod utnyttjar jämförelsevis låg densitet av fettvävnad att skilja mellan larver med förändrade fettdepåer. Flytkraft baserad analys är ett värdefullt verktyg för snabb, reproducerbar och ekonomisk screening.

En Flytkraft-baserad metod för att bestämma fettnivåer i<em&gt; Drosophila</em

Drosophila melanogaster is a key experimental system in the study of fat regulation. Numerous techniques currently exist to measure levels of stored fat ...

The overall goal of this assay is to determine any changes in fat storage levels that may be caused by a change in genetics, diet, or treatment. These methods can help answer key questions in the field of metabolism such as genetic or neuronal contributions to obesity predisposition. The main advantage of this technique is that it's fast, economical, and very reproducible when compared to other methods currently used.Individuals new to this method or new to working with wandering stage larva may struggle with this protocol because the developmental timeline of different genotypes can be highly variable. To begin this procedure, add a small dollop of yeast paste to the middle of a grate plate. Smooth down any rough edges with a spatula.Next, transfer the flies of interest into an egg collection chamber, and place the grate plate with yeast paste on top facing inwards. Then tape the grate plate to the egg collection chamber to keep it from falling out. Make sure to write important genotype and date information on the bottom of the grate plate.Subsequently, upend the egg collection chamber to allow the flies to lay eggs on the grate plate. After that, cover the egg collection chamber with an open box and place the covered egg collection chamber in an incubator at 25 degrees celsius for four to six hours to allow the flies to lay eggs. When it is done, take the grate plate out of the egg collection chamber and transfer the flies back into their original bottle of food.Use a spatula to wipe off any excess yeast paste from the grate plate. Subsequently, store the grate plate in a larger Petri dish in a humidified incubator at 25 degrees celsius for 24 hours. In this procedure, score the top layer of the food by plunging it with a spatula several times to allow the larvae to burrow and feed.22 to 24 hours after egg collection use a small paintbrush to collect 50 first instar larvae of the same approximate size and place the larvae in the experimental food vial. It's very important that larvae of the same approximate size are selected and transferred to the experimental food. This will ensure tight coordination of larval development.Next, clean the paintbrush with a laboratory wipe and ensure that there are no remaining larvae on the paintbrush before continuing to another genotype. Afterward, place the vial of larvae in an incubator at 25 degrees celsius and allow them to develop. Remove the vial of larvae from the incubator once there are 20 to 40 larvae wandering up the sides of the vial.Record the date and time of the assay as well as the number of wandering larvae. Then prepare the experimental solution by adding 11.5 milliliters of PBS and nine milliliters of 20%sucrose to a 50 milliliter conical tube. Add 20%sucrose to the vial until it reaches one inch from the top of the vial.Use a spatula to gently stir the food top layer to free the larvae that are still burrowing in it. All the larvae will rise to the surface of the sucrose solution. Afterward, gently transfer the larvae into the initial sucrose solution and gently stir them.While transferring the larvae to the experimental solution, be careful not to squeeze or puncture the larvae. This may alter the results of the assay. Cap the conical tube and upend it several times to thoroughly mix the solution.Then uncap the conical tube and swirl it to create a gentle vortex. Allow two to five minutes for the larvae to settle, either floating to the top of the solution or sinking to the bottom. Record the number of floating larvae, and the specific sucrose concentration tested.Then add one milliliter of 20%sucrose to the experimental solution to increase its density. Subsequently, use a spatula to stir the larvae gently. Following this, cap the conical tube and upend it several times to thoroughly mix the solution.Subsequently, uncap the conical tube and swirl to create a gentle vortex. Record the number of floating larvae and the new concentration. Continue this process until at least 95%of the larvae are floating.Add 20%sucrose to the conical vial until it is one inch from the top. Next collect all the larvae in a dissection dish filled with PBS. Examine the larvae under a microscope for any small larvae, prepupae, pupae, or any other differences between genotypes to ensure that the procedures were done appropriately.Then record the total number of larvae. Afterward, transfer 10 larvae on a laboratory wipe for drying. Label a microcentrifuge tube and place the larvae in the tube.Subsequently, flash freeze the larvae in liquid nitrogen and store the tube at minus 20 degrees celsius. Shown here is an example of how the buoyancy-based assay can detect both higher and lower fat stored in the genetically manipulated larvae. This figure shows that excitation or silencing of a certain subset of neurons in the larval brain induces lower and higher stored fat respectively.The same genetic background control was used in both cases. Here is an example of how this phenotype obtained by the density assay is corroborated by neutral lipid extraction and quantification by GCMS. Once mastered, this technique can be done within 30 minutes if properly performed, and may be performed in tandem with other genotypes.While attempting this procedure, it's important to remember to synchronize the development of the experimental larvae as much as possible. Following this procedure, larvae can be snap frozen in liquid nitrogen to be used in other methods like gas chromatography mass spectrometry in order to obtain additional information like the specific classes of lipid types in the larvae. After its development, this technique paved the way for researchers in metabolism to explore the genetic and or neuronal contributions to obesity in Drosophila.After watching this video, you should have a good understanding of how to quickly screen genotypes for changes in levels of stored fats.

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A Buoyancy-based Method of Determining Fat Levels in Drosophila

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Visually Mediated Odor Tracking During Flight in Drosophila

Visuellt Medierad Lukt Tracking under Flygning i Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

In-vivo Centrifugation of Drosophila Embryos

In-vivo Centrifugation of Drosophila Embryos

In vivo Centrifugering av Drosophila embryon

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

Biokemisk och hög kapacitet Mikroskopisk bedömning av fettmassa i<em&gt; Caenorhabditis elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

En förbättrad metod för noggrann och snabb mätning av Flight Performance i<em&gt; Drosophila</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Mätning av hemsyntesen nivåer i däggdjursceller

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

Ett förfarande för Permeabilization av<em&gt; Drosophila</em&gt; Embryon för analyser av Small Molecule aktivitet

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Kvantitativ Immunofluorescens analys för att mäta variationen i proteinnivåer vid Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Bestämma membranprotein Topologi Använda Fluorescens Proteas Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...