Gel loading Question - (Oct/22/2007 )
I have a quick question about sample buffer and reducing agent.
My sample buffer is 4x and reducing agent is 10x. Now, if I need to load 7.4ul of my sample in order to get the desired amount of protein loaded per well how much sample buffer and reducing agent do I add?
This is what I think I should do:
Sample buffer: 1.85ul
Reducing agent: 0.74ul
Total loading volume: 9.99ul
Does that look right?
Not really. When you calculate the amount of sample buffer and reducing reagent buffer to add in, you have to make sure that their FINAL concentration is 1x (usually the 1x is the working concentration). That means, you have to take in account the FINAL volume, in addition to the volume of your sample. For example, if you have 20ul sample and 5x sample buffer, the amount of sample buffer you have to add in is NOT 4ul (as in one fifth of the 20ul sample) but 5ul (so that it would be 5ul of 5x buffer in total 25ul (20+5) --> 1x working concentration of sample buffer at the FINAL count). In your case, for accurate but easy calculation, I would get 12ul as final volume (sample + buffer + reducing reagent). In that case, you need 3ul of 4x buffer, 1.2ul of 10x reducing reagent, 7.4 ul sample and top up with 0.4ul of whatever buffer that you keep your sample in (or you can use 7.8ul sample instead, if it does not make much different for your desired result). However, I have to ask, why you sample buffer and reducing reagent are separated? Say, if it is for protein gel, my sample buffer would already get the reducing reagent inside. But maybe you are doing something else? Just curious.