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:
  • 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 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.
  • 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).

    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.

     

  • 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.
  • Data selection:
  • 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 weights.
  • 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.
  • Quantitation:
  • 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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