how much do you load on the gel? - do you really dilute to certain OD? (Mar/07/2006 )
another stupid question but im thinking...im estimating the amount of protein after inducing with IPTG and boiling in sample buffer....in the molecular cloning it says "load 0.15 OD samples" so i was diluting my samples to 0.15 OD before loading....but that is just so faint...i barely see my lane...so Ill just try like 3ul or 5 ul of sample...how much do you load?
it's not a general rule,
I induces when OD600 reaches 0.5, and I induce one hour (toxic proteins).
If I want to see on coomassie blue, I load the equivalent of 10 uL of culture.
If proteins are well overexpressed (you see it on coomassie stained gel), I load 1 uL for western-blot, and adapt depending on the result.
It's "by chance"
I often compare before and after IPTG (induce at OD600=0.6). I centrifugate 250µl, dissolve the pellet in 80µl of sample buffer and load 5-10µl on the SDS-page. So it is nearly the same like Missele, from the original culture it is between 15 and 30 µl.
Missele and Ms-Olli, thank you so so much ...i loaded 3 ul and got very nice bands... ...thanx a lot again...have a great day all!!!!