A report on an experiment I did of doing electrophoresis with proteins

But for a fixed protein if m 0 and its corresponding A and n are known then one can treat the result as a standardization
such that it can now be used to predict masses of other samples of the same protein from their staining
intensities. But the prediction can have two different forms as follows,
• Like for standardization with BSA where m 0 is known, given an unknown sample of stain intensity I its mass
can be predicted to be
m =
2
m 0
“ ” 1
A n
I −1
1
• Like for standardization with Actin where m 0 is not known, given two samples of proteins of stain intensities
I 1 and I 2 one can predict the ratio of the masses of the proteins as,
17
m 1
m 2
= 2
“
A
I 1
” 1
n
−
“
” 1
A n
I 2
K. Some findings/observations of my own
• I started filtering the destaining solution twice to ensure greater efficieny of the chemical on reuse.
• I devised this method of transferring the gel on the scanner by using plastic supports while keeping it always
submeged in the destaining solution. This practically eliminates the risk of air bubbles getting trapped.
• The image analysis technique described here is of my own and I have not found that in any reference.
• I wondered if the staining/destaining step could be avoided by sending into the gel, protein mixed with Coomassie
Blue. But then this does not work as I found out that Coomassie Blue happens to have higher mobility than
the proteins and hence inside the gel it moves faster ahead without causing any staining effect.
III.
SOME THEORETICAL BACKGROUND
The entire physics and chemistry of the process of electrophoresis is a very complicated mix of phenomenon and
much of the numbers like the amount of each chemical to be used is very hard to rationalize by pure theoretical
calculation and are obtained by experimental optimization.
A. A basic overview of the idea
One of the key ideas behind electrophoresis is that if 2 solutions of different pH and different densities containing
charged molecules of different mobility can be stabilized against convective instabilities then the boundary between
the two fluids can be robustly maintained in an electric field. In that case the two fluids can move maintaining the
boundary or if the boundary is somehow made rigid (like through polymerization) then the charged ions can flow
through it in a predictable sequence without disturbing the boundary. This predictability of the sequence gives the
quite essential “stacking” effect in gel electrophoresis which holds the key to identification of the different protein
components in the initial solution.
The convective instabilities are generally avoided by either having the two fluids to be immiscible and of different
densities or by polymerization like here so that convection is prohibited simply due to structural rigidity. If the
boundary is moveable then the crucial observation is that inspite of the difference in mobilities of the mobile ions
on the two sides the velocities at which they move have to be the same since otherwise a gap gets created at the
interface through which electricity cannot conduct. Since in most situations the electric field and the mobilities are
given constants the formation of the gap is prevented by the rigid polymer matrix, the system attains this dynamic
quilibrium by dissociating the “correct” number of ions on either side. Eventually it wll be seen that whether the
system can dissociate to this optimal extent depends on whether the experimentalist has chosen the right dissociation
reactions and pH.
And much of these optimal values have been established over years of standardization of the experiment and only
some order of magnitude esimates can be gotten from pure theoretical arguments.