Sodium Azide 2.5 % 200 pi
Chloramphenicol 50 m g/m l in 50 % ethanol 80 pi Gentamicin Sulphate 10 m g/m l 400 pi Kallikrein Inactivator 20000 U /m l 25 pi
Table 2.2-2 Preservative cocktail for blood collection.
Stock Solution V o lu m e/5 0 m l Plasma
Benzamidine 1 M 50 pi
PMSF 0.2 M in anhydrous methanol (-20 °C) 250 pi Table 2 .2 - 3 Preservative solutions for lipoproteins.
2 .2 .3 Se q u e n t i a l Pr e p a r a t i v e Ul t r a c e n t r i f u g a t i o n.
2.2.3.1 Preparation o f Sodium Bromide Density Solutions. Base detisity solution (1.006g j ml).
57.0 g anhydrous NaCl, 0.5 g ED TA (Sodium salt), 5 ml 1 M N aO H ,
0.5 g Sodium azide.
Make up to 5 litres. Then add 15 ml distilled H^O.
This solution gives a refractive
All densit}^ solutions were prepared by adding a known weight o f solid sodium bromide to the base density solution. For accurate density determination, the refractive index value for each solution was measured using a refractometer (Bellingham & Stanley Ltd.). Fine adjustment o f densities were achieved by adding solutions, d = 1 . 2 1 or 1.478
g /m l (to fdensity) and d = 1.006 g/m l (to i density). 53
( g /m lf
Refractive Index Valdai Added -îUæiNx;
(approx. g /m l) V 1.006 1.3345 - 1.019 1.3368 - 1.045 1.3413 0.052 1.065 1.3448 0.075 1 . 1 0 1.3508 0.150 1.125 1.3547 0.165 1.157 1.3620 0 . 2 1 1.182 1.3643 0.25 1 . 2 1 1.3693 0.296 1.478 1.4160 0.79
NB. If turbid filter before final ad|ustment.
Table 2.2-4 Preparation o f stock density solutions.
2.2.3.2 Ultracentrifugation.
Bulk lipoprotein fractions were prepared in a 12 x 38.5 ml rotor (Kontron T F l' 50.38) either in a K ontron Centrikon T-2060 ultracentrifuge or a Sorvall OTD-65B ultracentrifuge.
2.2.3.3 Adjustment o f Plasma Density.
The following equation was used to calculate the volume o f density solution required to float lipoproteins:
Vi.dj + V^.dz = (Vi + V2).dj
W-Tere: = Volume o f plasma (or ultrafiltrate) V2 = Volume o f density solution to be added
di = Density o f plasma (or ultrafiltrate) dz = Density solution used for adjustment d^ = Density required
Sa?7îple Calculation:
A.djustment o f density fo r chylomicron, V L D L and ID L isolation.
Thus, for a typical isolation in a 29 ml centrifuge tube: Vj = 19.333 ml, V2 = 9.667
ml, dj = 1.006 g/m l, d2= U nknow n, and d^ = 1.019 g/m l.
(19.333 X 1.006) + 9.667 x d ^ = 29 x 1.019 19.448998 + 9.667dg = 29.551
9.667dz = 10.102002 dj = 1.045 g /m l
2.2.3.4 Chylomicron. VLDL and ID L Preparation.
Thus, 19.333 ml o f plasma was placed in a screw-capped polycarbonate centrifuge tube and its density adjusted to 1.019 g /m l by adding 9.667 ml o f 1.045 g /m l density solution. The samples were centrifuged for 20 h at 37000 rpm (105000 ^ and 16 °C. The lipoprotein fraction that floated to the top o f the tube was collected in less than 4 ml, using a syringe and needle. The fraction was then either dialysed for immediate use or washed. The washing step involves refloating the lipoproteins at their density o f isolation. Therefore, for this fraction, the lipoproteins were diluted to 29 ml with the stock 1.019 g /m l density solution and recentrifiiged.
2.2.3.5 L D L Preparation.
T he solution below the chylomicron, V LDL and ID L fraction was removed and discarded such th at 19.333 ml remained in the centrifuge tube. Its density was adjusted to 1.065 g /m l by adding 9.667 ml o f 1.157 g /m l density solution and mixed. The samples were again centrifuged for 20 h at 37000 rpm (105000 ^ and 16 °C. The L D L fraction was collected and dialysed or washed (in 1.065 g/m l solution).
2.2.3.6 H D L Preparation.
A fter
LDL
isolation,HDL
was prepared either as totalHDL
orHDL2/HDL3
sub- fractions. Alternatively,HDL
can be prepared from plasma after precipitation o f the apoB-containing lipoproteins followed by a single ultracentrifugation step.Total H D L Preparation.
The solution below the L D L fraction was adjusted to 19.333 ml and its density was adjusted to 1 . 2 1 g /m l by adding 9.667 ml o f 1.478 g /m l density solution and mixed. The
samples were centrifuged for 40 h at 37000 rpm (105000 ^ and 16 °C. The H D L fraction was collected and dialysed or washed (in 1 . 2 1 g /m l solution).
The solution below the L D L fraction was adjusted to 19.333 ml and its density was adjusted to 1.125 g /m l by adding 3.29 ml o f 1.478 g /m l density solution and 6.38 ml o f 1.125 g /m l solution. The isolation was perform ed as outlined for total HDL.
H D L . Preparation.
The solution below the HDL^ fraction was removed and discarded such that 19.333 ml remained in the centrifuge tube. Its density was adjusted to 1.21 g/m l by adding 6.13 ml o f 1.478 g /m l density solution and 3.54 ml o f 1 . 2 1 g /m l solution. The isolation was
perform ed as outlined for total H DL.
Preparation of H D L Follomnp Mamesium ! Phosphotungstic A cid Precipitation.
O ne hundred pi o f 0.5 M MgClj and 100 pi o f 4 % (w/v) phosphotungstic acid (in 0.19 M NaO H) were added to every 1 ml o f fresh plasma and mixed well. The plasma was then centrifuged immediately for 20 min at 2000 g and 20 °C. This step precipitated all apoB-containing lipoproteins, leaving plasma with