A laboratory exercise to illustrate protein–membrane interactions

A laboratory exercise to illustrate protein–membrane interactions

The laboratory protocol presented here takes about 3 hours to perform and investigates protein and lipid interactions. Students first purify His6‐tagged human apolipoprotein A‐I (apoA‐I) with Ni‐NTA affinity resin in a simple batch protocol and prepare multilamellar vesicles (MLV) from pre‐dried pho...

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Bibliographic Details
Published in:Biochemistry and molecular biology education Vol. 44; no. 1; pp. 86 - 94
Main Authors: Weers, Paul M.M., Prenner, Elmar J., Curic, Spomenka, Lohmeier‐Vogel, Elke M.
Format: Journal Article
Language:English
Published: United States Wiley-Blackwell 01-01-2016
Wiley Subscription Services, Inc
Subjects:
Apolipoprotein A-I - metabolism
apolipoprotein A‐I
Biochemistry
Biochemistry - education
College students
Electrophoresis, Polyacrylamide Gel
Humans
Laboratories
Laboratory Experiments
Laboratory Procedures
Lipid Bilayers
Lipids
Membranes
Molecular Biology
multilamellar vesicles
nanodiscs
Nanostructures
Pedagogy
Protein Binding
Proteins
Science Activities
Science education
Science Instruction
nanodiscs
apolipoprotein A-I
multilamellar vesicles
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Summary:The laboratory protocol presented here takes about 3 hours to perform and investigates protein and lipid interactions. Students first purify His6‐tagged human apolipoprotein A‐I (apoA‐I) with Ni‐NTA affinity resin in a simple batch protocol and prepare multilamellar vesicles (MLV) from pre‐dried phospholipid films. When apoA‐I is added to the MLV, much smaller protein/lipid nanodisc complexes are formed in some instances. Nanodisc formation can be monitored by a decrease in light‐scattering intensity at 340 nm using a simple spectrophotometer. Students will observe nanodisc formation with MLV formed from the anionic phospholipid dimyristoylphosphatidyl glycerol, which pack poorly into lipid bilayers, but not with MLV formed from the zwitterionic phospholipid dimyristoyl phosphatidylcholine, which form stable bilayers. This laboratory exercise is accompanied by questions and exercises that enable students a deeper of the dimensions of apoA‐I and nanodiscs as well as the biological relevance of nanodisc formation in the process of reverse cholesterol transport. © 2015 by The International Union of Biochemistry and Molecular Biology, 44:86–94, 2016.
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ISSN:1470-8175
1539-3429
DOI:10.1002/bmb.20929