Lipoprotein Analysis -week 3
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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 characterization.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: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 analyses.
Purification process
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 of lipophorin.
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
Apoprotein ratio
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.Experimental protocol
Scanning
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.Further analysis should be done in the Biology instructional computing laboratory (or at home).
Image analysis
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.