In-Gel Digestion and Protein Indentification

In-gel digestion and protein identification enables proteins separated on SDS-PAGE gels to be identified on a band-by-band basis. This takes advantage of the ability of SDS-PAGE to resolve complex protein mixtures into less complex mixtures based on protein cross-section. It is not unusual for individual bands in a complex mixture, such as a lysate or homogenate to contain hundreds of proteins. Alternately, protein isoforms that result from truncations or alternate splicing, which resolve by SDS-PAGE, can be analyzed separately and regions of the protein that are present or absent in the different isoforms can be observed.

SDS-PAGE gels should be stained with Coomassie, silver, or SYPRO. Please acquire an image of the gel to be submitted along with the samples. Gel bands can be excised either by our staff or by the client. Avoid submitting large sections of gel, because these will need to be subdivided for efficient digestion. In-gel digestion is done with trypsin. Other proteases are not recommended. Generally speaking, if a band can be visualized with one of the above stains, we will be able to detect it by LC/MS/MS. This may not be true for very low molecular weight proteins (<~10kDa). When submitting samples, please provide the organism to be used for database searching.

(UW System Pricing)
In-gel digestion: For the first 3 bands the price is $96/band. For 4 and above the price is $77/band.
Sample cleanup by solid-phase extraction: Each digest must be desalted and cleaned up by SPE at $25.50 per band.
LC/MS/MS: In-gel digests are run by Orbitrap short gradient LC/MS/MS. The price is $205 each for the first 3 runs, then $175 for each additional run.