Hello everyone I’m a research assistant who do molecular lab I have experience in western blot many times but the problem is about formula for preparing tris buffer for prepared SDS gel.Because the traditional tris buffer preparation I used to prepared for stacking gel is 0.5M and then adjust to pH6.8, and for separating gel is 1.5M and then adjust to pH8.8. In the some formula at my lab use tris buffer in the same concentration 1.5M but different pH for preparing stacking and separating gel so my question started about the effect of them to protein when running western blot analysis.
Narumon Mahaamnad
Hey Hi better not use the same Tris for preparing stacking and resolving buffers,
a simple and clear explanation is mentioned below:
If you read the basic principle of Laemlli, you will know it.
Briefly, it is for you to acquire proteins of different molecular weights 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 play a very important 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 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 struggle along as best they can (they do move but with lower mobility than the chloride ions). '
Very well explained.
https://mullinslab.ucsf.edu/sds-page/
- Badges
- Science method