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rabbit-in
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15 posts [100%]
Freising Bayern

 DIGE versus iTRAQ. Which approach is more expensive?

Dear all,

I’m thinking about doing a non-gel based technique approach to identify cell wall proteins (most of them are basic and highly glycosilated). Because of these characteristics, proteins doesn’t go easily inside of isoelectric focusing gels, so we will lose a lot of such proteins in a 2D approach.

I’m thinking about using iTRAQ technology, but until this point I don’t know how high would be the cost for analyzing 3 control and 3 treated samples. Do someone of you know, if the cost of such non-gel based techniques are much higher than a usual DIGE experiment. I refer to the cost of chemicals.

Thank you for your support.

Bcrespo
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 Re: DIGE versus iTRAQ. Which approach is more expensive? (rabbit-in)

Hi Rabbit,

Think that wiht iTRAQ it is necessary to do technical replicates. With DIGE, lots of people don´t do technical replicates. But, of course, if you know that your proteins don´t have good behavior in gels, make gels is only waste your money!

Jose

dfried
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 Re: DIGE versus iTRAQ. Which approach is more expensive? (Bcrespo)

There are interesting trade-offs both ways. I haven't done a careful cost analysis, but I think that at the end of the day (with appropriate replicates & power for each, plus MS identification for DIGE), you're looking at about the same cost overall (of course, this assumes you already have a good MS for iTRAQ).

David

MichelleReeve
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64 posts [100%]
Ithaca NY

 

Are you just interested in protein identification, or are you comparing treatments? Why not use label-free quantitation?
mysg
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78 posts [100%]
Salt Lake City UT

 Re: DIGE versus iTRAQ. Which approach is more expensive? (rabbit-in)

How much glycoproteins can be resolved in current 2D? Even 150kDa proteins are hard, but I remember some glycoproteins in the ECM I know of, e.g. fibronectin, laminin, are 200-400kDa, and syndecans are huge. I remember glycoresidues need to be removed before IDing on 1D western blot, but these steps are compatible with 2D??

bokajsen
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 Re: (MichelleReeve)

I agree with Michelle Reeve. Label-free LC-MS/MS has become very reliable (many good studies are out there). If we are talking costs I would consider aksing around and find a core-lab (academic/commercial) that can do label-free LC-MS/MS for you at a good price. Of cause you are not going to get isotypes like in 2D DIGE and there are other drawbacks - but personally I dont understand all the hype around iTRAQ. Maybe somebody can fill me in? Label-free LC-MS/MS (spectral of peptide counting) is very good for relative quantification and typically gives better sensitivity than iTRAQ...
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