Scroll down on this page to see all of the steps of the Western blot tutorial, using the "Auxilia" HIV case as an example.

You previously opened the protein and antibody files for Auxilia Case A when you ran the ELISA, so these file names are still visible in the "Opened & processed" window. Drag this window towards the top of the Data Screen, just below the 8 buttons shown below. These 8 buttons correspond to the 8 wells on a protein electrophoresis gel.

1. Click the "Protein: protein HIV.txt" line the "Opened & processed" window to activate it, then click the "Load" button. Note that the button for well #1 turns blue, indicating that it has been loaded.

2. Click the second well to select it (indicated by the purple color), then click the "Load" button again, as shown below...

3. Repeat this process until you have loaded 5 of the 8 wells of the protein gel...

4. You now need to associate an antibody sample with each well. Click the first antibody line in the "Opened & processed" window ("ANTIBODY: anti youngest child.txt"), then click the number "1" as shown below. A blue outline appears around the number, indicating that the well is associated with this antibody.

5. Click the second antibody line ("ANTIBODY: anti Auxilia.txt"), then click the number "2" as shown below...

6. Repeat this process until each of the five wells is associated with one of the five antibody samples.

7. Click the "Run" button (shown below) to run the Western blot...

8. To label the Western blot, first hilight text in the "Loaded" window as shown below, click the "Copy" button...

...and then click thed "Paste" button, as shown below. Note that the hilighted text automatically appears in the label below the blot.

9. Repeat this process until all five lanes have been labeled (you can edit text directly in the label, as has been done in the example below)...

10. Once the lanes have been labeled, click the "Photo" button and select "1"...

and the photo will be taken and you will be automatically taken to the Photo Screen, part of which is shown below. Click the "Save" button on this screen as shown below and save the blot, using a unique filename. (This photo can later be uploaded if you decide to use the Case It Web Editor to build a web page of your results).

11 . Click the "Go back" button to go back to the Data Screen...

24. It is necessary to identify the proteins on this blot to determine if a person is positive for HIV. To do this, use the "Data" button as shown below. Select lane 5 because it is the positive control, and all proteins will be present.

25. When the above option is selected the protein weights in kilodaltons (kd) appear in the data matrix to the right (see below). Click the rightmost fragment, as shown below, and note that its weight is outlined in red in the data matrix, and also appears below the gel (see bottom of graphic below). This particular protein is called the "p24" protein (note that his number is close to the calculated value that the simulation generates).

26. Click on the fragment to the left of this fragment, and note that its weight (32.65) is outlined in blue in the data matrix and also appears below the gel. This is the "p31" protein.

27. By looking at the numbers above from left to right, you see the complete sequence of weights for these fragments. Here is a table relating these weights to the names of the fragments, for diagnostic purposes:

The latest criteria from the Center for Disease Control in Atlanta, Georgia is as follows:

Based on these criteria, how would you interpret the Western Blot below? Why does the ELISA differ? (remember, the ELISA is a relatively quick screening test that can result in false positives, whereas the Western blot is more definitive).