I have carried out a native PAGE with 4 reaction mixtures. To each I had added an equal volume of EDTA (1 µl/1mM) to sequester any divalent ions and an equal volume of calmodulin (5 µl/0.5 mg/ml). I added 2 µl of calcium (2 mM), magnesium (2 mM) and manganese (2 mM) ions respectively to three reaction mixtures and had a control that was made up to an equal volume with Tris-HCl. All reaction mixtures were made up to 10 µl with Tris-HCl. Here I ran into a problem with the waterbaths and the reactions couldn't be incubated at 37 degrees Celsius.
After I added the loading buffer to each, I added the 4 reaction mixtures to the polyacrylamide gel and electrophoresed for 45 minutes at 150 V. After this I placed the gel in Coomassie Blue stain for 20 minutes.
After the destaining process, I reviewed the gel to find that the calmodulin in the 4 lanes had migrated the same distance. I had expected the EDTA control to have migrated the furthest on the gel, with Ca2+ and Mn2+ migrating similar distances due to their similar ionic radii; making it a good substitute for the binding to the calmodulin. I was unsure of how far the Mg2+ would travel as I thought it would have a lower affinity to the calmodulin due to its binding to the N-terminus rather than the C-terminus. However, I was wondering if this was negligible in the absence of Ca2+, although now I can't be sure due to the results of the gel.
I was wondering what errors in the experimental design or process could have resulted in this? And if the experiment was run correctly, what results would be expected?
The first image is the result I was expecting. The second is the results from my gel electrophoresis.