Lipoprotein preparation
From LipidomicsWiki
Contents |
Enzymatic modification of LDL
E-LDL was generated under sterile conditions. LDL isolated from plasma of healthy blood donors was diluted to 2 mg/ml protein in PBS (w/o Ca2+, Mg2+). To 5 ml LDL diluted in PBS (2mg/ml) 80 µl Trypsin-EDTA Solution (1x) (sterile filtered with 0.5g porcine trypsin and 0.2g EDTA; 4Na/L HBBS; Sigma) and 50 µl cholesterol esterase (Seikagaku, Japan, 100U in 1ml sterile H2O) were added.
Subsequently the solution was incubated at 37°C for 24–48 h (solution is getting cloudy).
Important: Trypsin-EDTA solution has to be portioned into 100 µl and stored at -20°C not longer than 3 month. For every preparation freshly thawed trypsin has to be used.
References
Bhakdi S, et al. J Exp Med. 1995 Dec 1;182(6):1959-71.
Torzewski M et al. Arterioscler. Thromb. Vasc. Biol. 1998 Mar;18(3):369-78.
Kapinsky M et al. Arterioscler. Thromb. Vasc. Biol. 2001 Jun;21(6):1004-10.
Mildly Cu2+ oxidation of LDL
Mildly oxidized LDL was achieved by dialyzing purified LDL fractions (1 mg protein/ml) against 5 µM CuSO4 for 40 h. The oxidation process was stopped by dialysis in PBS/EDTA. Further extensive dialysis in PBS was made. Afterwards, the Ox-LDL was sterile filtered and protein content was determined by Lowry-method. The mildly oxidation of LDL was controlled by electrophoresis.
References
Wieland E et al. Proc. Natl. Acad. Sci. USA, 1993 Jul 1;90 (13):5929-33.
Kapinsky M et al. Arterioscler. Thromb. Vasc. Biol. 2001 Jun;21(6):1004-10.
Lipoxygenase modified LDL
Growth medium
450ml DMEM (low endotoxin) + 50ml serum + 5ml Gentamycin + 2.5ml G418 sulfate
Incubation medium
500ml DMEM (low endotoxin) + 5ml Gentamycin + 2.5ml G418 sulfate
Procedure
Grow C12 cell-line in growth medium until it is grown confluent. Remove serum and wash with sterile PBS.
Dissolve LDL to a concentration of 50 µl/ml in incubation medium and add 13ml of this mix to every flask. Incubate at 37°C for 18h. Harvest supernatant and filter through a 0.45 um filter.
Take Millipore concentration filter and moisten it with sterile PBS.
Concentrate the harvested mmLDL from 15ml to a volume of 2.5ml. Measure protein concentration and dilute to 50µl/ml. This is also the working concentration.
References
Sigari F et al., Arterioscler Thromb Vasc Biol. 1997 Dec;17(12):3639-45.
Benz DJ et al, The Journal of Biological Chemistry, Vol 270, No.10, Issue of March 10, pp. 5191-5197, 1995
Minimally modified LDL through long term storage
Minimally modified LDL was obtained by storage of LDL at 4°C for 3-6 month in plastic tubes.
References
Watson AD et al, J. Clin. Invest., Volume 95, February 1995, 774-782
Harkewicz R et al, Journal of Biological Chemistry Vol.283, No.16, pp.10241-10251, April 18, 2008.
HOCl-modified LDL
Bestimmung der NaOCl-Konzentration
dilute NaOCl (Aldrich) with 0.1M NaOH 1:400 and measure the extinction at 292nm.
calculation: mol/L = E x factor of dilution/350
350 = molar extinction coeffizient of NaOCl
Modification of LDL
Before the modification desalt proteins with Sephadex PD10-column. Equilibrrate the column with PBS (10mM, pH 7,4), give protein solution onto the column, fill up with PBS to 3ml and subsequently eluate the proteins with PBS (max 3 ml).
Incubate proteins (lipoproteins max. 2mg/ml) in PBS (10mM, pH 7,4) with NaOCl on ice: To maintain neutral pH neutral provide HCl (0,1M) (Empirical value: with 0,7M NaOCl double volume of NaOCl).
Additon of NaOCl (in the desired molare ratio oxidation agent : protein): If required dilute NaOCl in PBS, pipete a drop of NaOCl onto the wall of the tube and mix quickly with the protein solution. Subsequently incubate at least 30 min at 4°C.
References
Malle E et al. Arterioscler Thromb Vasc Biol. 1995;15:982-989.
Hazell Linda J et al. Biochem J 1993, 290, 165-172.
