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The iTRAQ reagents are the first set of multiplexed, amine-specific, stable-isotope reagents that can label all peptides in up to eight different biological samples enabling simultaneous identification and quantitation, both relative and absolute, while retaining important PTM information. There are two types of iTRAQ reagent kits, 4plex and 8plex. The iTRAQ reagents - 8plex kits are also available in more economical bulk packs.
Community Discussions »
Innovation in peptide-tagging chemistry
iTRAQ reagents are an isobaric, peptide-tagging technology that enable you to label all primary amines, regardless of peptide class. Because important information, such as post-translational modifications (PTMs), is retained, this tagging system allows you to extract more detailed information from your samples.
Figure 1: iTRAQ reagent - 8plex protein quantitation.
Higher-throughput protein biomarker discovery
Obtain protein identification and quantitation simultaneously for up to eight samples for higher throughput biomarker discovery. The straightforward iTRAQ reagent workflow has less rigorous chromatography demands than non-labeling approaches and provides high-confidence results. And contrary to label-free profiling approaches, iTRAQ reagents are completely compatible with fractionation strategies that allow you to "dig deeper" and evaluate lower-abundance proteins
Experimental flexibility with confident identifications
The ability to analyze up to eight different samples simultaneously gives you even more experimental-design flexibility. Multiplexing enables you to compare numerous sample states and provides the flexibility to run duplicates and triplicates. MS/MS fragmentation of peptides labeled with iTRAQ reagents - 8plex results in cleaner spectra with increased signal, giving higher confidence in peptide identifications. And the tagging and analysis of multiple peptides per protein further enhances protein identification.
Figure 2: iTRAQ reagents - 8plex protein identification.
Solutions for quantitative biology
Whether you are performing relative or absolute quantitation, iTRAQ reagents - 8plex, MS instrumentation, and application software provide an integrated solution with the ease, flexibility, and confidence in protein quantitation and identification you need for quantitative biology. These reagents are ideal for many applications including pull-downs, membrane protein studies, discovery and validation analysis for biomarker elucidation, and absolute quantitation of target proteins of interest. The reagents are compatible with most mass spectrometers but are optimized for SCIEX instruments.
Is iTRAQ available in bulk?
Yes. Bulk reagent is available in 25 and 50 unit kits of iTRAQ reagents only. You will need approximately one Buffer kit per 10 units.
Why is there a large difference in incubation times between 4-plex and 8-plex iTRAQ?
iTRAQ 8-plex is a different molecule than 4-plex, the reaction time is longer (120 minutes versus 60 minutes)
Are 114 -117 from 4-plex the same as 114 – 117 of 8-plex?
No, 4-plex and 8-plex are different reagents and should not be combined in an isobaric experiment
Why do I see a different charge state distribution of the target peptides after using iTRAQ?
Because of the different final pH, you may see a difference between the 2+ and 3+ charge states. A shift to more 2+ ions can usually be accomplished by adjusting the pH to 4.0 with 5mM ammonium acetate
Can I re-use the iTRAQ reagent after the vial has been opened?
No. Each vial of iTRAQ reagent is filled and capped under inert gas. The reagent is easily degraded by moisture leading to poor subsequent labeling efficiency. In the case of bulk reagent kits, there is a specific protocol to follow for aliquoting and storage.
My iTRAQ solution seems to be a different colour, is it OK?
Probably. The colour of the iTRAQ reagent can vary slightly depending on many factors ranging from trace metals to moisture. The reagent performance is typically unaffected.
How much protein can I label?
Each vial of 8-plex reagent contains 2 µmol of iTRAQ reagent. This is usually sufficient to label 20-100 µg of protein digest. For plasma and serum samples which contain many non-peptide primary amine compounds, the protocol may need to be adjusted. Important elements include buffering to pH 7.5 to 8.5, buffer concentration between 90 – 100 mM, iTRAQ reagent concentration at 40 mM (±5%), and total organic concentration greater than 65%
How long can I store sample after labelling?
we recommend you label only when you are going to analyze the samples immediately, please do not store your labelled samples for longer than two weeks.
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