Lipoprotein Analysis -week 3
During this week you will analyze the SDS PAGE and determine
the protein content in the lyophilized fraction of lipoproteins gravimetrically.
You will also extract the lipids from lipoproteins
according to Bligh and Dyer (1959) for further
In the next experiment (Lipid analysis),
lipid composition of the extract will be determined by thin layer chromatography
(TLC) followed by Gas liquid chromatography (GLC).
Analysis of gel:
From the stained gel, you will be able to get information about:
the separation during ultracentrifugation
the purity of your lipophorin fraction
the molecular weight of the apolipoproteins
the molar ratio of the three apoproteins in the lipophorin molecule
While qualitative evaluation can be done on the stained
gel itself, any quantitative analysis is best done on a scanned image of
the gel. Using appropriate software, you can then carry out the required
This should be obvious by visual inspection. You should see
three characteristic bands of apoLp-I, apoLp-II, and apoLp-III in your
pooled lipophorin fraction. Hopefully, these bands peak in the fractions
that you pooled. Other, adjacent fractions may contain these three bands,
but you should not see all of these at densities that are much higher.
Purity of lipophorin
From the scanned image, you can estimate the purity of your
pooled lipophorin fraction if you assume that all proteins are stained
by Coomassie Brilliant Blue with similar intensity (this is not necessarily
true, however). Dividing the densities of the three apoproteins by the
combined densities of all bands in that lane will result in the percentage
Molecular weight of apoproteins
The electrophoretic mobility of a protein in an SDS gel is equivalent to its molecular weight. Hence, we can determine the size of the three apolipophorin molecules if we generate a standard curve from known molecular weight markers. This is accomplished by plotting their Rf values against the log of their molecular weights (Mr).
distance between sample well and protein band
______________________________________ = Rf
distance between sample well and dye front
From a quantitative analysis of the gel scan, you can determine
the molar ratio between the apoproteins, again assuming that each binds
Coomassie Brilliant Blue similarly. The density of each bands is then proportional
to the amount of protein in it. In order to get information about the molar
ratio, you have to divide each density reading by the molecular weight
of the apoprotein, as determined before; the ratio between the resulting
values represents the molar ratio.
First you must scan the stained and destained gel. Place
the gel between two clear plastic sheets, and make sure that no destaining
solution leaks from the sandwich. Go to the Mac in the adjacent computer
lab and start the "DeskScan" software.
Pre-scan the gel as greyscale picture.
Select the area you need to scan (= the entire gel).
Scan the gel and save the file as a TIFF file.
Transfer the TIFF file to a disk.
Further analysis should be done in the Biology instructional
computing laboratory (or at home).
Gel analysis is best carried out with the NIH Image program.
This program was developed by the National Institute of Health. Information
about its use can be obtained from the NIH
Web site. Since the program is free, you can also download it onto your own Mac, if you have one. A free PC version of Image, called Scion Image for Windows, is available from Scion Corporation. (There is also Image/J , a Java program inspired by Image). For our analysis we need the program and the gel scanning module. This software is loaded onto the Macs in the Instructional Computing room.
Start the Image program.
Open your TIFF file from your disk. Detailed instruction
can be found in the Image manual.
If the Setup to plot Gel and Plot lane commands
are not shown in the Special menu, use the Load Macros to
open the macros contained in the file Gel Plotting Macros.
Close all image windows except for the gel to be analyzed.
Use the rectangular selection tool to outline the first lane.
This is the left most lane for vertically oriented lanes and the top lane
for horizontal lanes.
Select Mark First Lane in the Special menu. A copy of the
image will be displayed with the first lane outlined.
Move the rectangular selection (by clicking inside it and
dragging) and outline (using Mark Next Lane ) each of the other
lanes in succession. Select the Marker lane, the pooled lipophorin lane,
and one or two of your lipophorin containing fractions.
Use Plot Lanes to generate the lane profile plots.
Molecular weight determination:
In the marker lane, identify the major bands and their molecular
Measure the distance for each band.
Draw a semi log plot of molecular weights vs relative mobility (Rf) for the marker lanes. Use this estimate the molecular weights of prominent bands.
Use the line drawing tool to draw base lines and drop lines
so that each peak defines a closed area as shown above. Note that you can
hold the shift key down to constrain lines to be vertical.
Measure the areas of the peaks by clicking inside each one
in succession with the wand tool.
Option-click with the text tool to automatically label the
peaks, in reverse order, with the area measurements. The area measurements
are also recorded in tabular form, and can be displayed (Show Results)
printed (Print) or exported (Export) to a spreadsheet.
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