Membrane Lipids 1Phospholipid 2Glycolipid 3Cholesterol Classifying Lipids Phospholipids

Membrane Lipids 1)Phospholipid 2)Glycolipid 3)Cholesterol

Classifying Lipids

Phospholipids 2011 Beate Fuchs et al

Glycolipids : Sugar containing lipid Cholesterol : Steroid

Lipid extraction • Folch method • Bligh and Dyer method • Matyash method (modified version of Folch method, Bligh and Dyer method) • Etc.

Saponification • Saponification is a process that produces soap, usually from fats and oil. It involves a reaction between a base, usually sodium hydroxide (caustic soda), and an ester group on a compound. Triglycerides are an example, which is an ester of a fatty acid. The triglycerides are hydrolyzed to form the sodium salt of a carboxylate. In addition to soap, such traditional saponification processes produce glycerol. "Saponifiable substances" are those that can be converted into soap.

Thin Layer Chromatography(TLC) Absorbent sheet is a thin layer of fine silica powder spread uniformly over an inert support such as a sheet of glass or plastic. (TLC는 보통 극성인 Silica plate를 사용하고, 전개용매로는 비극성 용 매를 사용한다. ) Adsorption chromatography separates lipids of different polarity

• The Order of migration of lipids during chromatography depends primarily on polarity. • The less polar the lipid, the greater its affinity for the nonpolar solvent (compare the polar silica gel) and hence the greater the migration.

Lipid structure formula <cholesterol> 386. 7 g/mol <sphingomyelin> 731. 60 g/mol

Phosphatidylcholine (786. 11 g/mol) Phosphatidylethanolamine (744. 03 g/mol)

PROCEDURE • Lipid Extraction from mammalian cells 1) 100Φ Plate의 Medium 제거. 2) Trypsin-EDTA (TE buffer) 1 ml을 이용하여 plate에서 cell pellet을 얻은 후 1. 5 ml tube로 옮겨준다. 3) 1, 200~2000 rpm에서 5분간 원심 분리하고, 상층액은 제거하여 cell pellet을 얻는다. 4) Ice-cold PBS 1 ml로 pellet을 풀어준 후 1, 200~2, 000 rpm을 풀어 준 후 1, 200~2, 000 rpm에서 5분간 원심 분리 5) Pellet을 피해서 조심스럽게 상층액을 제거한다. 6) 얻어낸 pellet에 Chloroform : Me. OH (1: 2) 혼합액 (400 ul)을 넣고 Vortex 7) 동량의 Chloroform (400 ul) 을 넣고 다시 Vortex 8) 동량의 D. W. (400 ul) 를 넣고 다시 Vortex 9) 3500 rpm에서 10분간 원심분리 10) 하층 (Chloroform)층을 조심스럽게 덜어 낸다

PROCEDURE • TLC 전개 1) Overnight으로 흄후드에서 말린다. 2) 20 ul 전개 용매에 녹인다. Lipid extract 3) Cholesterol(10 mg/ml), Phosphatidyl ethanolamine dioleoyl(10 mg/ml), Phosphatidyl choline(10 mg/ml), Sphingomyelin(10 mg/ml), FAME mix, Lipid extract(12)결과물)를 3 ul씩 TLC 판에 점적한다. 4) 용매가 들어있는 Tank에 넣고 2. 5 -3 hours 전개한다. TLC 판

Result of lipid extraction

Result {Assume adsorbent more polar than solvent} 1. Which band moved the fastest? 2. Which band has the relatively most polar molecules? 3. Which band had greater affinity for the mobile phase? 1 2 3 4 5 6 1 : Cholestrerol / 2 : PE / 3 : PC / 4 : Spingomyelin / 5 : Lipid extract / 6 : FAME mix Chloroform : Methanol : Acetic Acid : D. W = 100 : 70 : 20

PROCEDURE • Saponification 1) lipid 에 0. 5 M Na. OH (90 % Me. OH)을 700 ul을 넣는다. 2) 70 ℃ Heat Block에서 한 시간 가량 녹여준다. 3) Con. HCl을 100 ul 넣는다 4) Centrifuge 3500 rpm 5) Crude soap, Na. Cl 확인

Result of saponification 중화반응 과정에서 생긴 Na. Cl 이 염석 효과를 일으켜 crude soap를 침전시키게 합니다. Me. OH and DW contain Contaminant Lipid(crude soap), Na. Cl