This video will show you how to operate the densitometer
When using the densitometer, make sure to fit the scanner with the lid that has a white background
instead of the lid that has a glass background.
This will ensure a clearer image of the gel
When handling the gel, make sure to wear gloves to prevent your hands from getting stained.
Gently remove the gel from the petri dish and place it down onto the scanner.
Avoid trapping in air bubbles when doing this.
with your gel loaded onto the scanner, open up the “Labscan” software on the computer
Click on the “scanner icon with a question mark” to obtain a preview of your gel.
Once the preview appears, use the “crop tool” to draw a rectangle around your gel in order to crop out the bottom.
Make sure you do not cut out any bands or lanes.
Next, click on the “scanner icon with no question mark”.
An enlarged image of your cropped gel will appear.
Click the floppy disc icon to save your gel image.
Make sure to save it as a Tiff file.
Name the picture with your group number along with SDS or IEF depending on the type of gel you used,
and save your image in the designated folder.
The gel images need to be converted in order for the analysis program to easily detect the bands.
To do so, open the IQTL calibration converter.
Click on “add file” and select the image file you saved.
Click on “convert file” and a notification will appear after the conversion is complete.
To analyze the image of the gel, click on the “Image Quant TL” software.
Click on “1D gel analysis”.
Select your converted photo.
The file name of the converted photo should be the original file name followed by “(IQTL)”.
Open the file.
To select and edit the lanes manually, click on the “stepwise” icon from the left panel.
Then select the “manual” icon.
Go to the parameters tab located on the bottom of the left panel
and identify the “number of lanes” present on your gel you wish to select.
Proceed to click and drag a box across the image of your gel,
ensuring that each lane is fitted with a rectangular outline.
You can always click the clear button and reselect if you are unhappy with the lane selection.
To edit the lanes, 2 options are available: edit single lane and edit multiple lanes
To edit individual lanes, select edit single lanes from the drop down menu.
Select the move button to move the rectangular outline side to side in order to better fit the gel image.
Make sure to not move the outline up or down to avoid accidentally cutting off bands.
Select the bend/resize button to trim off the sides of the bands.
This produces better quality peaks for analysis.
For lanes that are slanted, left click on the white square tie point to drag and fit the outlines diagonally.
Left click on the centre of each lane to add a white square tie point.
Bend and stretch the outlines by left clicking on the tie point and dragging it to fit distorted gels.
To remove tie points, right click on them.
If you want to edit all lanes at once, select edit multiple lanes from the drop down menu.
When you are happy with your lane positioning, click next.
Click on the parameter tab again.
Select the image rectangle option.
Draw a rectangle on the blank area of any selected lanes.
This will be your background color that will be subtracted from the color of your bands.
Make sure the peaks shown on the graph are all above your subtraction line.
When finished, click on “subtract” and then “next”.
Click on the “detect” icon and all bands on your picture should be outlined.
if there is a blank area mistakenly selected as a band, right click in the middle of the band to delete it.
If there are any bands thats not detected automatically, left click in the middle of the band to add it in.
You can also adjust the width of the bands, but it’s not necessary for the purpose of this experiment.
The width of the bands are used for protein expression analysis
where the colour density of the bands are used to determine protein concentration.
However, the objective of this experiment is to only identify the proteins based on their molecular weight.
When you finish selecting your bands, click on the next button
Click on the “parameter” tab again.
Select “SDS” or “IEF” according to the type of gel you are using.
The molecular weight of SDS and IEF standards should be included in those options respectively.
Select the standard lane by left clicking on it.
Remember the standard lane should have nine bands in total.
click on the parameter tab again,
and from the dropdown menu select “linear log curve” option.
Click compute and a graph showing the standard versus the distance travelled on the gel will appear.
There should be a window on the bottom of your screen showing all data values.
Make sure you click on the “all lanes” tab before proceeding to the next step.
Click “edit” on the top of the screen, and then select “export to excel”.
An excel sheet with all your data will appear automatically.
Save your excel file, your original gel image and your converted gel image with the bands identified to you USB for your report.