| | Re: equal loading on western blot?
here is what I do:
collect samples, store at -80
on the day of SDS-PAGE, I defrost the aliquotes for protein assay and do the assay (note-all diff conc.)
then i do my calculations on how much volume of each sample I need to give me a bit more protein than I'm going to load in a well (say I load 15ug, so I take volume tat gives me 20)
then I add ice cold (from freezer) acetone at approx x10 the volume, spin at 13000rpm for 10 min at 4C (this is to precipitate protein, and separate some of the crap that dissolves in acetone)
the I aspirate or pour off the acetone and let prot. pellets dry (if you leave acetone, stuff will float and not sink into the well)
than add my cracker buffer (1% SDS and other stuff including B-mercaptoethanol and bromophenol blue) and resuspend pellet. Amount added will depend on the amount of protein in the pellet, so that I have between 0.5 and 1 ug prot per uL cracker buffer.
Then I denature proteins at 95C for 3 min
And load into the wells. Now, I have prepared more solution more than I'm actually loading so that I don't have to scrape every drop out of the tube to make it even loading. This way I get the same amount of protein and same volume into each well.
I think if you have too much protein per uL SDS, it may not coat the protein properly hence running time will be wrong. Having said that, I had a few mistakes when I miscalculated and had x50 the amount of protein per uL of cracker buffer and although the gel looked totally overloaded, all bands were in the correct places.
Hope this helps