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Separating protein isoforms and quantifying activity


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#1 SweDennis

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Posted 22 July 2011 - 01:16 AM

Dear colleagues,

I would very much like advice on an issue. I am working with glutamine synthetase (GS) proteins in barley. There is one cytosolic GS protein and one slightly different chloroplastic isoform, and most likely one more cytosolic isoform. I am using a synthetase activity assay for measuring total GS activity in my root and shoot samples. However, I would like to be able to separate the activities of the different isoforms, and preferably high-throughput for many samples. Does anyone have a suggestion on which method to use to separate them?

The GS isoforms are slightly different in size and pI. I am mainly considering ion exchange chromatography (IEC), capillary electrophoresis (CE) or native PAGE, for which we have equipment in our lab. But some questions arise:
- Can IEC be made high-throughput, quick and cheap?
- How big is the protein yield loss in IEC?
- Can the sample volume in CE be big enough to allow a subsequent enzymatic activity assay?
- Is it in any way possible to recover the enzymes for activity measurement after running them through a native PAGE?

I will very much appreciate any comment on this topic.
Cheers,
Dennis

#2 mdfenko

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Posted 22 July 2011 - 06:22 AM

The GS isoforms are slightly different in size and pI. I am mainly considering ion exchange chromatography (IEC), capillary electrophoresis (CE) or native PAGE, for which we have equipment in our lab. But some questions arise:
- Can IEC be made high-throughput, quick and cheap?
- How big is the protein yield loss in IEC?
- Can the sample volume in CE be big enough to allow a subsequent enzymatic activity assay?
- Is it in any way possible to recover the enzymes for activity measurement after running them through a native PAGE?

iec can be performed on large amounts of material. the cost will be determined by the matrix you choose. it does not have to be performed on hplc or fplc equipment if you pack your own classical lc columns and use classical gradient formers.

yield is variable depending on conditions of the run, lability of the protein, etc. but, the yield will be consistent as long as conditions are maintained.

ce uses nanoliter (or lower) volumes of sample. it would take many runs collected into the same vial to obtain a usable amount of protein.

there are a few ways to recover usable protein from native page. one way that we used to use is to stain a strip from the gel, line it up, cut out the band area from the unstained portion and homogenize in a suitable buffer, spin out the acrylamide mash and use the supernate. you can also electroelute from the gel slice. but homogenizing is faster.
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#3 SweDennis

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Posted 27 July 2011 - 10:51 PM

iec can be performed on large amounts of material. the cost will be determined by the matrix you choose. it does not have to be performed on hplc or fplc equipment if you pack your own classical lc columns and use classical gradient formers.

yield is variable depending on conditions of the run, lability of the protein, etc. but, the yield will be consistent as long as conditions are maintained.

ce uses nanoliter (or lower) volumes of sample. it would take many runs collected into the same vial to obtain a usable amount of protein.

there are a few ways to recover usable protein from native page. one way that we used to use is to stain a strip from the gel, line it up, cut out the band area from the unstained portion and homogenize in a suitable buffer, spin out the acrylamide mash and use the supernate. you can also electroelute from the gel slice. but homogenizing is faster.


Thanks for your reply. I may try this method then with native PAGE. What will determine the buffer condition, apart from the properties of my enzyme?
cheers,
Dennis

#4 mdfenko

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Posted 28 July 2011 - 12:47 PM

I may try this method then with native PAGE. What will determine the buffer condition, apart from the properties of my enzyme?

the formulation of the native page. there are a number of buffer systems and pHs in which the systems operate. you find the one most compatible with your protein or devise one yourself (or modify an existing system).

you can also add various components to stabilize your protein (other than salt).
talent does what it can
genius does what it must
i do what i get paid to do




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