Generally we use 1.5M Tris (pH=8.8) for preparation of resolving gel but 1.0M Tris (pH=6.8) for stacking gel. How does Tris having two different pH in a single polyacrylamide gel help to resolve protein bands? What is the exact chemistry behind this? Thanks to all of you in advance.
Hi,
If you read the basic principle of Laemlli, you will know it.
Briefly, its for you to acquire proteins of different molecular weight essentially running at the same time which is why pH needs to be different. Three pH, stacker, resolving and the running buffer inclusive of glycine playing a very imp. role.
see the explanation here:
'Glycine is a weak acid and it can exist in either of two states, an uncharged zwitterion, or a charged glycinate anion (that is to say, negatively charged). At low pH it is protonated and thus uncharged. At higher pH it is negatively charged. When the power goes on the glycine ions in the running buffer want to move away from the cathode (the negative electrode) so they head toward the sample and the stacking gel. The pH there is low and so they lose a lot of their charge and slow down. Meanwhile, in the stacker and sample the highly mobile chloride ions (which are also negatively charged) move away from the cathode too. This creates a narrow zone of very low conductance (in other words very high electrical resistance) in the top of the stacking gel. Because V=IR almost all of the voltage that you put across the gel (110 Volts is typical for stacking) is concentrated in this small zone. The very high field strength makes the negatively charged proteins move forward. The trick, however, is that they can never outrun the chloride ions. If they did they would find themselves in a region of high conductance and very low field strength and would immediately slow down. The result is that all the proteins move through the stacker in a tight band just behind the moving front of chloride ions. Behind them, the pokey glycine ions straggle along as best they can (they do move, but with lower mobility than the chloride ions). '
Very well explained.
http://mullinslab.ucsf.edu/Protocols%20HTML/SDS_PAGE_protocol.htm
Hi,
If you read the basic principle of Laemlli, you will know it.
Briefly, its for you to acquire proteins of different molecular weight essentially running at the same time which is why pH needs to be different. Three pH, stacker, resolving and the running buffer inclusive of glycine playing a very imp. role.
see the explanation here:
'Glycine is a weak acid and it can exist in either of two states, an uncharged zwitterion, or a charged glycinate anion (that is to say, negatively charged). At low pH it is protonated and thus uncharged. At higher pH it is negatively charged. When the power goes on the glycine ions in the running buffer want to move away from the cathode (the negative electrode) so they head toward the sample and the stacking gel. The pH there is low and so they lose a lot of their charge and slow down. Meanwhile, in the stacker and sample the highly mobile chloride ions (which are also negatively charged) move away from the cathode too. This creates a narrow zone of very low conductance (in other words very high electrical resistance) in the top of the stacking gel. Because V=IR almost all of the voltage that you put across the gel (110 Volts is typical for stacking) is concentrated in this small zone. The very high field strength makes the negatively charged proteins move forward. The trick, however, is that they can never outrun the chloride ions. If they did they would find themselves in a region of high conductance and very low field strength and would immediately slow down. The result is that all the proteins move through the stacker in a tight band just behind the moving front of chloride ions. Behind them, the pokey glycine ions straggle along as best they can (they do move, but with lower mobility than the chloride ions). '
Very well explained.
http://mullinslab.ucsf.edu/Protocols%20HTML/SDS_PAGE_protocol.htm
The process mentioned b all colleagues above is named: isotachophoresis.
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