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Interpretation & Troubleshooting on SDS/Native PAGE

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Interpretation & Troubleshooting on SDS/Native PAGE

Postby schroddycat » Sat Dec 15, 2012 4:12 pm

Dear All,

I'm currently doing purification on the glutamate decarboxylase. After running both DEAE and gel filtration purification, I did both SDS and Native PAGE to verify my results. For SDS PAGE, there were 3 faint bands found and all of it are very near to each other. For Native PAGE, there was only 1 band and it was very near to the loading well. My questions are:

1. How do I interpret the results of PAGE and determine whether my purification is done? If native PAGE has only 1 band whereas SDS has 3, does it mean I've got my enzyme purified already?

2. As the band of native PAGE is very near to the loading well, I suspect it wasn't just my enzyme but it probably is a protein aggregate. How do I verify this i.e. is it only one protein or protein aggregate? If it's protein aggregate, how do I improve my native PAGE?

SDS PAGE

Image

Native PAGE

Image
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Postby JackBean » Sun Dec 16, 2012 10:17 pm

The Native PAGE looks really bad, it probably didn't really enter the gel. What percentage was the gel?

The bands on SDS-PAGE are pretty faint. How does your sample look now on gel exclusion chromatography?

What was source of your protein? I guess it wasn't native enzyme, because then you could hardly have only three bands (unless you've loaded very low amount of proteins, with which would correspond the faintness of the bands).
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