Biology Reference
In-Depth Information
rotors for this step since LLDs tend to stick to the wall of the centrifuge tube and thus
cause cross-contamination in the various gradient fractions. Immediately after centri-
fugation, the bottom of the tube is carefully punctured with a fine needle and fractions
are collected into collection tubes. Alternatively, a tube slicer can be used to collect
fractions and minimize carry over from other fractions. Two milliliters per fraction
are collected for a total of six fractions, labeled fractions #1-6, with #6 being the
top (most buoyant) fraction. The fractions, together with aliquots from the various
steps of preparation, are then used for analysis for their biochemical compositions
and physical prosperities. The following are examples of analyses we performed.
7.2.2.7
Sample analysis
7.2.2.7.1
Analysis of protein composition by SDS-PAGE and
immunoblotting
After purification, samples should first be analyzed for recovery and contamination
by immunoblotting. We use ER-resident proteins such as PDI as markers for recov-
ery of ER lumenal content, and immunoblot for apoB to monitor the degree of con-
tamination of LLDs with apoB-containing VLDL precursors. SDS-PAGE and
immunoblotting procedures are similar to standard procedure used for most proteins;
however, it is important that 2-5% SDS (final concentration) should be included to
dissolve lipids. The sample buffer we use for immunoblotting of LLD-associated
proteins is listed in
Section 1
. Due to the low abundance of LLDs and large volume
collected from the density gradient, fractions #4-6 are concentrated 15-fold using
centrifugation-based protein concentrators (Pierce) with molecular weight cutoff
of 10 kDa. By comparing proteins enriched in each fraction obtained from the gra-
dient with aliquots saved during the preparation, fractions #4-6 are LLD fractions
with distinct associated proteins. An ER lumenal lipase, Ces3/TGH, can be used
as a marker for fraction #4 (corresponding to smaller LLDs), and apoE for fraction
#6 (corresponding to larger LLDs).
Fractions can be analyzed for protein and lipid compositions using established
methodologies described elsewhere.
7.2.2.7.2
Analysis of LD particle size by FPLC and native PAGE
These analyses are performed in combination with protein and lipid composition
analysis. Information obtained from these analyses is useful not only to understand
the physical property of LLDs, but more importantly, as a read out for protein and
lipid compositions associated with the various subsets of LLDs. This information is
especially beneficial when comparing between different experimental conditions,
such as genetic backgrounds, nutritional states, or drug treatments.
Gel filtration chromatography.
Combined LLD fractions are concentrated and ap-
plied to a Superose 6 size exclusion FPLC column (Pharmacia, Uppsala, Sweden). Elu-
tion of various TG-rich LLD subfractions is determined by on-line detections of TG
content. Specifically, eluted fractions are mixed in-line with the Infinity
TM
Triglycer-
ide Reagent (Thermo Fisher Scientific, Inc., Waltham, MA) using a post-column
T-connector/Solvent Delivery Module (model 110B, Beckman Coulter, Mississauga,
