I have minimal protein concentration and would like to add the lowest possible amount of loading buffer and reducing agent. We usually use 10x reducing agent and 4x loading buffer. Therefore, if one has to load 19.5uL of lysate, than one would add 3uL of reducing agent and 7.5uL of loading buffer. BUT, what if one has to load say ~ 30uL of lysate to get a reasonable concentration, what do you recommend as the minimum amount of reducing agent and loading buffer?
If you prepare your own loading buffer and reducing agent, you can make them more concentrated than the commercial preparations, resulting in less dilution. If you use a thicker gel (1.5 mm instead of 1 mm), you can load 1.5 times more sample into a well of the same width.
So if you ended up with ~45 uL of load volume (30 uL sample volume) and the well volume is ~40 uL max then leaving the cap open during ~80C heating of microfuge tubes for about 10 min. will allow sufficient evaporation to reach a reasonable load volume.
Hello Ayman
In reality you can actually load half the amount of loading buffer to save on such volume:
As long as the SAME concentration of loading buffer is added to samples and also EMPTY WELLS then you will be fine.
The main problem as long as you have requisite components in your buffers (which still exist at 1/2 x; Remember these volumes are not arrived at from first principles; This isn't atomic physics !!) and as long as those ion concentrations are uniform across your gel, so no perturbation exists, which would result in an irregular electric field then polyacrylamide gel electrophoresis will proceed as normal
Hi, I guess by using thick combs, wells will be more spacious to hold the more sample. I think you can still reduce the loading buffer to half.
This link will really help you,
http://instanano.com/characterization/theoretical/reducing-agent-calculator-nanoparticles-synthesis/
- Badges
- Science method