Gradient mixing - (Nov/28/2008 )
How does gradients like pH gradients used for isoelectric focusing or sucrose gradients used for centrifugation (rate-zonal or equilibrium) not mix?
Say for example the pH gradient used for isoelectric focusing. I know they are made from carrier lymphocytes and such but doesn’t mixing occur at the boundary between the two pH zones. Since pH is determined by the exchange of protons between species, why doesn’t the lower higher pH region donate some of the protons to the lower pH and therefore mess up the sharp boundary between the pH gradients?
In the density gradients used for centrifugation, why doesn’t sucrose from the more dense (more concentrated sucrose region) move into the less concentrated sucrose region?
I don’t understand the theory behind it, if someone could explain that would be great. Thanks.
Say for example the pH gradient used for isoelectric focusing. I know they are made from carrier lymphocytes and such but doesn’t mixing occur at the boundary between the two pH zones. Since pH is determined by the exchange of protons between species, why doesn’t the lower higher pH region donate some of the protons to the lower pH and therefore mess up the sharp boundary between the pH gradients?
In the density gradients used for centrifugation, why doesn’t sucrose from the more dense (more concentrated sucrose region) move into the less concentrated sucrose region?
I don’t understand the theory behind it, if someone could explain that would be great. Thanks.
in ief, the pH gradient is formed by the migration of ampholytes while under the influence of the electric field (immobilized gradients are poured and maintained because they are attached to the matrix). when the electric field is turned off they will start to mix but it takes a while to be noticeable.
sucrose density gradients will equalize if left standing for a long period.