We used pH6.8 in stacking and pH8.8 in resolving gel. In the class, the professor explained that the glycine change is like:

glycine+ <-------> +glycine- <------> glycine- 

it is said that most of the form will be +glycine- and glycine-. Why? And why does it change the charge when it runs from the stacking gel to resolving gel? What is the role of glycine in the running buffer for SDS-polyacrylamide gel electrophoresis?

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    $\begingroup$ In your original query I believe that you are confusing/conflating the electrophoresis with the transfer to a membrane. The electrophoresis is abbreviated SDS-PAGE. After the electrophoresis is complete you have several choices, including: fixation of the proteins in the gel followed by staining to visualize the protein bands; autoradiography if any of the proteins are radiolabelled; or transfer to a membrane if you have an antibody that recognizes the protein of interest. I have edited the post to correct this. I believe you are asking about the composition of he Tris-glycine buffer. $\endgroup$ – mdperry Nov 22 '15 at 22:25
  • $\begingroup$ Gly is an amino acid its charge depends on the pH. It is probably ladder component, but I am not an expert of this. gmwgroup.harvard.edu/pubs/pdf/951.pdf $\endgroup$ – inf3rno Nov 23 '15 at 19:23

The pH of stacking and resolving gel are set in such a way that one is above and one is below the pI of gly (5.97).Therefore, Gly has two different charges in stacking and resolving gel. That is in stacking gel gly has no charge (which will allow the protein to stack according to size in this part of the gel rather than separating because of presence of gly in between the protein molecules, constraint in separation of proteins) but in resolving gel charge is negative since the pH is higher (constraint in movement of proteins no longer present so separation of protein based on size possible).

Hope this was helpful..

  • $\begingroup$ Could you add a reference or link to allow for background reading? $\endgroup$ – AliceD Nov 28 '15 at 18:29

since the stacking gel have a ph of 6.8 the glycine will attain a neutral charge(by the isoelectric point and ph relation)thus the chloride ions travel faster followed by the sample and then at the last glycine ions,thereby stacking the sample in between both.when it reaches the resolving gel the ph increases which gives glycine a negative charge.so they travel faster along with the chloride ions leaving back the sample to get seperated without any hinderence

  • $\begingroup$ Welcome to Bio. Could you add sources to your answer? $\endgroup$ – AliceD Oct 30 '17 at 16:22

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