Tandem mass spectroscopy protein analysis

Sample form and amount
We accept bands or spots that are clearly visible with Coomassie blue and that are cut out from regular 1D or 2D SDS-PAGE gels. Amounts of 10-50 ng for average size proteins is the low end of the amount requirements. Therefore, all visible Coomassie-stained bands contain enough material for reliable analysis. We recommend to avoid silver staining since it sometimes interferes with sample extraction from the gel. We have been successful with sypro orange dye.

Note: Dried gels We now accept protein bands that are cut out from dried, cellophane covered old gels that were stored at room temperature (up to 15 years old).

Edman degradation sequencing

Sample amount
We require a minimum 20 pmol of blotted protein. 50 pmoles is very comfortable and generate unambiguous data.

Sample form
We accept PVDF blots or purified lyophilised/solubilised proteins.

Blotted onto a PVDF membrane (preferred)
The protein should be electroblotted from 1D or 2D gels and stained with Coomassie Blue (R250). Following staining/destaining, blotted membranes should be rinsed thoroughly with deionized water. The sample should be as concentrated as possible (15 to 20 pmol / lane) and several gel lanes may be loaded and submitted. The whole membrane may be submitted with a copy showing the band(s) of interest or the bands may be cut out and submitted, please indicate band molecular weight.

Protein in solution or lyophilised
30 to 150 µl of solution (H2O, Acetonitrile, Propanol). Pure lyophilised protein is also acceptable.
Protein should be free of detergent, salts and buffers. Please note that any amine-containing chemical present (e.g. Tris) will impair the adduction step.
Extra charge applies for this type of sample as we will run pre-purification at nominal fee to ensure complete removal of amine-containing impurities.

Sample purity
PVDF blots: well separated bands.
Liquid: Single purified proteins. Immunoprecipitation and affinity-column purified usually give good samples for sequencing.
In any case, a mandatory additional column purification step is applied to all incoming solution samples.