Hi, I've been trying to run a Western blot for a number of months and they are just not working out. I'm trying to probe for HIF1(alpha), and I'm using a 4-20% TG gel. The DNA was taken from mouse brains that were sonicated in RIPA buffer. Usually the first two lanes turn out fine, but the next few lanes ALWAYS run weird. They either make a hill shape, where the lane is narrower than it should be and the middle is raised up further than the ends, or they are simply narrower than the first two lanes. Any thoughts on why this would be? We have tried different boxes, different antibodies, and different types of gels (BT with MOPS). The pictures are really bad quality but it gives an idea of what I mean; it makes it impossible to quantify these Westerns.
One common reason for lanes to run oddly on SDS-PAGE is the composition of the sample: too much detergent or lipid or salt, or the pH is wrong. Since these are brain extracts, it might be that there is too much lipid. Try using a smaller amount of the sample in the maximum possible volume of SDS-PAGE sample buffer. Obviously, this will reduce the sensitivity of your Western blot, hopefully not by too much to be useful.
One common reason for lanes to run oddly on SDS-PAGE is the composition of the sample: too much detergent or lipid or salt, or the pH is wrong. Since these are brain extracts, it might be that there is too much lipid. Try using a smaller amount of the sample in the maximum possible volume of SDS-PAGE sample buffer. Obviously, this will reduce the sensitivity of your Western blot, hopefully not by too much to be useful.
Hi Elyse, I agree with Adam. Brain tissue (by mass) is mostly lipids.
High concentrations of endogenous lipids interfere with SDS-PAGE because it relies on SDS (a strong anionic detergent) binding to proteins and making them equally charged. Polyacrylamide retards mobility of big proteins in PAGE compared to smaller proteins.
Anyway, you could minimize effects of endogenous lipids in your sample . You have some options. Add more SDS to your samples. Use acetone precipitates of homogenized brain tissues. Use triton X-114 phase separation of lipids and membrane proteins from soluble proteins in brain homogenates.
Thank you both! I will try some of your suggestions and see if it helps. I appreciate it!
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