Separating AA via electrophoresis...what pH to use?

[labqi]

Hey guys! I hope studying is going well...

I had a quick question or two regarding AA separation. I was doing a Kaplan Section Test...and the question was:
the isoelectric points of glycine and lysine are: 6.0 and 9.6 respectively. These amino acids can be separated by electrophoresis at what pH.
A) 6.0 B)9.6 C)Both and A&B

I had originally picked 9.6. At this point the lysine would be a neutrally charged (ps. would the sc still be charged), and glycine i believe would be negatively charged thus moving toward the anode. This logic applies to A as well. But when I was doing the Berkeley Review, it said to use the higher pI (I think it was an acidic aa and basic amino acid). Am I missing something, why isn't it B only though based on what I 'learned' or thought I learned from Berkeley.

Also, if the AA is positively charged it tends to migrate toward the cathode. But isn't the cathode the area where they are loaded? So why would they move? I just can't visualize where the cathode and anode are in respect to the loading area.

Thanks in advance!

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[labqi]

Bump

 

[Czarcasm]

So the main discrepancy here isn't necessarily your line of thought with regard to the charge on the amino acids. The real issue here is determining which charge we would prefer to have to travel to the anode or cathode. For electrophoresis, the anode is (+) and the cathode is (-). This is a convention that confused me as well, but reading up on it, I believe it had something to do with the fact that the gel is situated between a discharging capacitor (not entirely sure). Based on that convention though, the amino acids get loaded into the docking area on the cathode side and will travel to the positive terminal (anode). This is the usual convention, but it's possible TBR may have switched it up to really make sure you're understanding what's really happening.

As far as the charges themselves: when pH is above the pI, it's negatively charged. when pH is below the pI, it's positively charged. And when the pH=pI, it's neutral. Because we're trying to separate the amino acids, we want one of them to be neutral and the other to be charged. Determining which charge you need for the amino acids to travel is ultimately the key piece of info that you need to answer this question correctly.

 

[dudewheresmymd]

As far as the charges themselves: when pH is above the pI, it's negatively charged. when pH is below the pI, it's positively charged. And when the pH=pI, it's neutral. Because we're trying to separate the amino acids, we want one of them to be neutral and the other to be charged.

if ph > pI are all 20 amino acids negatively charged?

 

[Czarcasm]

if ph > pI are all 20 amino acids negatively charged?

Yes.

 

[dudewheresmymd]

Yes.

and this applies for polypeptides as well as single amino acids?

 

[Czarcasm]

and this applies for polypeptides as well as single amino acids?

Yes.

 

[dudewheresmymd]

thanks!

 

[dudewheresmymd]

Yes.

for acidic amino acids, at ph> pi the charge would be -2 and for basic amino acids if ph > pi, the charge would be -1 right?