5 replies to this topic

[schroddycat]

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




Native PAGE

[mdfenko]

a little more information is required.

what are the acrylamide percentages in the sds and native page.

pH of the native page and pI of the protein of interest.

molecular weight of the protein of interest and subunits, if known.

the limited mobility in native page could be due to aggregation, size of whole molecule, charge at the running pH, acrylamide concentration...

the multiple bands in sds could be caused by multiple proteins purifying together, degradation, heterologous subunits of the native protein, contamination, artifacts...

according to worthington, glutamate decarboxylase from e. coliis a hexamer of ~50kDa subunits (total 310kDa). the version from your source may be similar.

[schroddycat]

Thanks mdfenko. In SDS and native PAGE, the acrylamide % is 30. Resolving gel is 12%.

pH of the native page is 8.3 pI of my enzyme is not known but from other papers, GAD is acidic enzyme and should be below 6. As it is newly isolated enzyme, the MW of enzyme or its subunit is yet to be known. I have run another SDS PAGE with another newly purified sample and its result is still the same. How do I know it is exactly purified yet? But from the 3 bands as highlighted, I still need to add one more step to purify further right?

I think of running native PAGE to get more ideas about the enzyme but I need the solve migration problem first. Question is how?

Edited by schroddycat, 19 December 2012 - 06:58 AM.

[mdfenko]

12% is too restrictive for a protein that may be ~300kDa (for the native gel) i would use a 7% gel.

can you identify the standards?

did you include blank samples (protein buffer with sample loading buffer) in the blank lanes? some of the bands you see may be from an artifact caused by keratins in dust (~50-70kDa).

[schroddycat]

Thanks mdfenko for your feedback. I thought I would come back again but I need to do further tests first. I haven't try using low %gel first but I proceeded with CTAB PAGE because my GAD enzyme was mentioned to be acidic enzymes by various papers, with optimum pH around 5.5.

In CTAB PAGE, I didn't see the faintest band at all along my protein sample. I used silver staining and that should give me indication even if the enzyme concentration was minuscule. But there wasn't any band at all and one of the sample lane has had some smears. Protein ladders were fine. I really don't know what's going on but I'm pretty sure there's at least some protein in my sample somehow.

Any advise on this or I should perform another Native PAGE with lower % of gel?

[mdfenko]

pH optimum would be for enzyme activity. pI would be useful to determine what type of native gel to run (acidic, neutral, basic).

7-7.5% acrylamide used to be considered pretty much standard for electrophoresis. for higher molecular weight proteins 4-5% would be used.

i can't give advice on ctab-page because it may be one of the few page methods with which i have no familiarity (i am familiar with the use of ctab for transfer).

i would try native page with a lower running gel concentration and maybe add a 3-3.5% stacking gel (stacking gel should have a different pH buffer than the running gel).