loading buffer and western blot help! - (Oct/18/2010 )

hi,
i would like to ask stupid question about the way you prepare the lammeli buffer. My way is the folowing, i have stock of loadinge dye 3x: (-20°C). when i do sds-page gel, i just add my sample to loading dye, boil and load. on the other hand, i made sds page in another lab, and someone noticed that, im wrong, and i should add water to prepare my sample with loading dye! ex; if i want to load 50ug of protein from 10mg/ml of total extract and 32ul as final volume: i take 4.55ul, then, i add 10.6ul of loading buffer and then rest 16.85ul of water. this is correct but im not used to do that before!
what i do, is i take the volume of protein according to the concentration i want to load then, i add directly loading buffer 3x about 7ul, and i boil, and load!!
can this make difference in the result??
please, help, i know that it looks stupid, but, i need to understand, if i have to add water always??

You should always work such that your loading buffer is going to be at a 1x concentration when you load it onto the gel... i.e. if you have a 3x buffer, and 9ul of sample, you would add 3ul of loading buffer, mix, boil, etc., and load the whole lot onto the gel.

Edited to add: If the loading buffer is at the wrong concentration (too high or too low) when running, it can affect how your proteins migrate and/or the appearance of the lanes.

wouldn't that be 4.5ul 3x loading buffer for 9ul sample?

Sorry, quite right, I was thinking 9 ul total volume.

hi, thanks for your replies!
but, where is water in all this?
im wondering if adding water to fill until total volume before boiling is commun for you or not??
thanks

the way the dilution is presented here is without the use of water.