I have been observing double bands for protein molecular marker(especially 15 kDa band) every time I am doing SDS-PAGE. Sometimes even the samples appear to have a double band. These are 12.5% handmade gels and sample running voltage is 160 V. Can anyone suggest how to resolve this issue.
Thank you for your answers.
Yes I have separated a protein that appear on the gel as a double band.
You need to separate your protein with 8 or 10percent gel so you can observe the double band.
And be careful as the proteins run very fast in low percentage gel, so slow down the voltage.
Few questions and ideas to consider:
1) Have you run titrated protein samples to see what is the best concentration to run?
2) Have you tried running your samples on a gradient gel to allow for better protein separation? Perhaps, 5-20% or 3-18%?
3) I'm thinking since you only have double bands for specific sized proteins in the ladder, perhaps those proteins may have degraded over time or through multiple freeze-thaw cycles. Try using a fresh sample to see if you are still seeing double bands. If yes, then make sure you aliquot your protein ladder into smaller volumes to avoid the freeze-thaw cycles.
Good luck!
Thank you all for the valuable suggestions.
Meera J. Patel I haven't tried the titration or the gradient gel approach as I just need to check the purity of the sample before moving on to the next step of the experiments. To answer your third point; Yes, I too had the same thing in mind. So I ordered a new ladder; but I am still getting the double band.
Sara Kishta Mohamed The size of the protein of interest is ~14 kDa. So, I and not using low % gels. But, Yes I will probably try lower volt to Run the gels.
Venketsubbu Ramasubbu Okay.
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