Hi all,
I just isolated some protein from my neutrophils yesterday and ran this SDS-PAGE today. For reference, I made a 15% gel recipe (as my protein, LC3B, is very light) and ran the gel at 80V for 30 min and 120V for one hour. I loaded 40ug of protein per well (which came out to be 28 uL), and 5 uL of protein ladder.
As you can see, my bands seem to be "spilling over" into other wells and my mark lanes are not straight or defined. I have been having this problem for a long time and I am wonder why? Any suggestions would be greatly appreciated.
Dear Stefan Michael Sampy
It seems that the loading of protein samples per well is too high, which starts pushing the neighbouring lanes (which also have too much load), thus the migration of the proteins occurs horizontally to accommodate the high load of the proteins. Notice that your marker has shrunk up after a while. I would suggest you to load less amount of proteins. In addition, 28 ul of protein sample per lane is too much. 20 ul should be the limit for proper loading. The high volume of sample may cause diffusion as well.
I am sharing with you one of my SDS PAGE result where I experienced similar situation.
All the best.
Satyendra Mondal Thank you for the suggestion! I tried the PAGE again except this time, I loaded 20uL and ran the gel at 70V. The bands are still curving slightly but to a lesser extent so next time I will try 15 uL.
Stefan Michael Sampy did you check the temperature of the gel while running? It can also occurs due to overheating of the buffer which increases with time. If overheating is an issue, lower the volt/current or and place the apparatus on ice and or run in a cold room.
Satyendra Mondal yes, I was thinking that overheating could be an issue because I an running the PAGE at room temperature. However, my lab colleagues who run their gels at even higher voltages (120 V for instance) don't experience this problem so temperature does not seem to be an issue.
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