I have plasma from knockout mice treated with vehicle or raloxifene/tamoxifen. Looking for a small acute phase protein (~12 kDa). I first did a 1:1 dilution of sample to laemmli buffer, but the signal is saturated. I decreased the amount of protein loaded to 15 μg which comes out to a loading volume between 1 and 2 μL. I cannot load less because I would be loading less than 1μL. How should I go about diluting my sample? What should I use to dilute the plasma? How much should I dilute by?
I suggest to make an intermediate stock (dilute 10x your original samples with PBS, ex: 1ul sample+9 ul PBS), Then it would be easy to load your desire protein concentration.
Hi there,
If it's just about gel loading then dilute your sample further in Laemmli buffer so that you can load a significant volume onto the well for your 15µg of protein.
As suggested by Ahmed Dhamad, prepare stock solution first by diluting 10x, 15x, or whatever times with your lysis/working buffer.... and load from that stock.
As others suggested, step-wise dillutions. But, also, keep in mind to vortex/agitate WELL between each dilution and quickly sample and load gels (no wait times!) is best if you have enough sample, keeping each dilution step in a tube just in case to go back. Then, verify by running those dilution steps through Western blot gel.
As you do this, also dilute a loading control in same fashion. There should be consistent variation downward to higher dilutions on the verification gel you run.
Hope this helps.
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