Sample handling instructions

When running SDS-PAGE gels, precast gels are best, but if you are creating you own, please let the gels set overnight before running them, as this prevents further modifications happening such as crosslinking. Never use commercial instant staining kits as these are incompatible with downstream MS analyses (however we have seen a report that apparently Imperial Stain from Pierce is compatible). Also gel stains increase background signals in MS, so please only use as much as is necessary to visualise your protein. Do not use a microwave to speed up staining, as this will permanently fix the protein in to the gel.

Contamination can arise from a variety of sources. When scanning/imaging the gel ensure that the gel is in plastic, and do not use overhead transparencies. Keratin is everywhere, and will taint everything. In higher concentrations it causes severe problems as during an MS cycle only the top n peaks are selected for MSMS analysis, and so keratin peptides can ‘out-compete’ real analyte peptides. So please use gloves and process gel bands under a fume hood, and a clean working environment – but no soap, and always use fresh solvents! Polymers must be absolutely avoided as they easily ionise and can subsequently suppress peptide signals, also they cause considerable – and often irreparable - harm to columns. Please avoid ‘low-binding’ eppendorfs, and never use parafilm. Avoid detergents over 2-3% in concentration

In Short, Please...

Contact us BEFORE you start your project

Provide us with as much information about your sample / protein as you can (use sample submission form)

Use clean containers to stain & store gels, Label them "only for MS!"

Use a lid for these container (contaminations! we belive they can fly)

Wear gloves during sample handling, everytime

Cut gel bands on a clean bench & a clean glass plate using a clean (=new) scalpel knife

DON'T use parafilm at all

DON'T bring in samples just before your weekend

DON'T use commercial kits for staining

DON'T apply microwaves to expedite coomassie staining

DON'T use overhead transparencies to scan gels

DON'T use detergents

DON'T use >5% glycerol

For intact Proteins,...

we will need at least 10 µL of a protein at a concentration of >1 mg/ml

Samples need to contain ≤ 5% glycerol

Please avoid usage of ony detergent

Proteins should be as pure as possible

Simple mixtures of a few proteins might work

A mixture of a modified and a non-modified version of a protein migth work

Please sent us a gel picture - we will evaluate, if your sample is pure enough