FAQs
↓ Q: How do I submit a sample?
A: Please contact us via email using the following adress: ms-service@zmbh.uni-heidelberg.de
↓Q: What buffer should my sample be in when submitted?
A: We prefer the samples to be already submitted as Coomassie-stained SDS-PAGE gels. The gels should be thouroughly destained in water and delivered in a clean & keratin-free container. Alternatively samples can also be submited in Laemmli buffer and will be run on pre-cast gels provided by us (+20€ extra charge/per gel). Sample for 1D electrophoresis should not exceed a volume of 50 µl, for 2D electrophoresis the maximum is 125 µl (not exceeding 50 ug of total protein).
On request and depending on the project needs we can also perform in-solution digestion employing either the widely used “8M Urea protocol” or the “FASP protocol” (see Methods - Sample preparation). For this, please contact us in advance for details regarding sample buffer composition, interfering substances etc. !
↓ Q: Are detergents in my sample a problem? How can I remove them?
A: Most standard detergents used in the laboratory (e.g.: SDS, Tween, Triton, ...) are NOT compatible with downstream LC/MS-analysis and thus should not be contained in your samples. Therefore, if they cannot be ommited during sample preparation they have to be removed. For this, we recommend to precipitate your protein(s) using the “Wessel-Fluegge protocol” (see Protocols). The resulting dried protein pellet can then be processed by us.
Alternatively, one could also use the MS-compatible detergent RapiGest SF sold by Waters Corporation. For details regarding this option please contact us.
↓ Q: How much protein is necessary for identification or quantification?
A: In general Coomassie stained gel bands have a high success rate for identification. We recommend to load up to 50 ug of proteins per well for a complex sample mixture (total amount) and a minimum of 50 ng of a single protein band.
↓ Q: Can I submit silver stained gels?
A: Generally, Silver-staining is not the best method to combine with mass spectrometry. We rather recommend using sensitive colloidal Coomassie blue stains. But note that shortly developed silver stained gels, for which mass spec compatible reagents were used, can also be submitted. For this, the SilverQuest Silver staining kit from ThermoFisher Scientific (Cat.nr: LC6070) is preferred.
↓ Q: Is a specific 1D gel system or Coomassie-stain recommended?
A: Most SDS-PAGE systems are suitable for downstream mass spectrometry. We can recommend 4-12% gradient NuPAGE Bis-Tris precast gels (ThermoFisher Scientific preferred) as they offer excellent separation and sensitivity together with a colloidal Coomassie blue stain (ThermoFisher Scientific or SERVA Quick Stain preferred).
↓ Q: When can I expect my results back?
A: We process the samples according to “first come first serve” and aim for data return within two weeks. However this might vary, depending on the total sample number in the queue as well as the complexity of the workflow. Thus, an individual approximation of the processing time will be given during sample submission.
↓ Q: What possibilities are there for quantification?
A: Possibilities include label free (MaxQuant LFQ, Spectral counting), chemical (e.g. Dimethyl) or metabolic labeling (e.g. SILAC) as well as absolute labeling using AQUA peptides.
↓ Q: What are the first steps for SILAC? Where do I get the labeling media?
A: Cellular isotope labeling has to be done by you, the user. The required SILAC media can be obtained from various suppliers e.g. Silantes, ThermoFisher Scientific etc.. To obtain reliable SILAC-quantifications we highly recomend to check the incorporation efficiency of the heavy amino acids within your cell line of choice before starting any the real experiments. Note that we have successfully performed SILAC on E.coli, yeast and various mammalian cell lines like HEK-293, HeLa and microglial cells.
↓ Q: What do you charge?
A: You can find our current price list under "Protocols". For a more specific quote adapted to your project, please contact us by email.