Hey guys! I hope everyone is well, I ran a protein western probing for LC3 antibody. When I image my gel the bands from my treated samples do not appear as straight lines, rather 'n' shaped bands (my control samples are ok).
To clarify, I made up my lysates, then ran two gels from the same lysates probing for two different antibodies. One gel ran perfectly and the bands could be visualised and imaged. I will upload images of both membranes.
Conditions: samples boiled for 6min at 90 degrees
4ul Reducing agent, 10ul SDS loading buffer.
44ul sampled loaded per well
Gel ran at 190volts
Any advice would be greatly appreciated
Thank you in advance
Mohammed
I have often observed this when the gels are a bit older. If you are pouring your own gel, I recommend you use freshly made gels. Otherwise check the date on the pre-made gels and order new.
Thank your for your response Brian. I am using pre cast gels from invitrogen, NuPage 10% Bis-Tris Gel 1.5mm
the mW for the protein target in the gel with the frowning bands is 17-18kDa and the mW of the bands in the seconds image is around 60kDa. Could the voltage affect the migration of the lower weighted proteins maybe? I used 190v
How did you prepare the cell lysates? by chemical? biological? or physical methods, such as sonication? Did you load the total cell lysates or the soluble fractions/ clarified lysates/?
How much did you load? Did you denature the proteins at 90-100 degrees for 5 to 10 minutes?
I do not think the voltage caused the problem since your control samples look good.
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