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Check List for preparing in-gel samples for Mass Spectrometry

____ Use sterile technique principles in preparing your samples, running gels and cutting out

bands or spots.

____ Gel bands or spots from Invitrogen NuPage gels produce better single to noise in the

mass spectrometer than BioRad Criterion gels.

____ Colloidal Coomassie Blue, Invitrogen Simply Blue or Protea Super Blue Ultra stains are preferred,

but any staining technique is acceptable if the technique is known to be mass spectrometry compatible.

If you do not know if you technique is MS compatible check with the vendor or with us.

If you use a silver stain kit, do NOT over stain the gel, even if it is MS compatible.

____ Capture an image of your gel to submit with your sample. You may use our gel

scanner to reduce the risk of contamination. Please contact Lauren DeVine at (This email address is being protected from spambots. You need JavaScript enabled to view it.) or call 410-614-6968.

____ Rinse natural color, non-coated eppendorf tube with HPLC grade methanol.

____ Place gel in clean container, washed glass or petri dish.

Do NOT use plastic wrap or parafilm, they are good sources of keratin.

____ Cut PROTEIN band or large spots into 2-5 pieces.

(pieces smaller than 1x1 mm will be sucked up into a pipette tip when dehydrated)

Cut out just the band or spot and the sooner the band or spot is cut out the better.

No extra unstained gel around the band or spot.

Stay away from the edges of the gel lane.

Large amounts of gel decreases efficiency of in-gel digestion and peptide extraction.

____ Place gel pieces into rinsed eppendorf tube.

If more than 8 samples, please put gel pieces in rinsed 96 well plate (Isc BioExpress 65120,

plates greiner, V-bottom) with a X-Pierce cover (Isc BioExpress T-2439-100).

____ Cut BLANK control gel band or large spots into 2 to 3 mm pieces.

____ Place gel pieces into DIFFERENT rinsed eppendorf tube.

If samples are in a 96 well plate, place samples in the following order: A1, B1, C1, etc.

____ Destain silver stained proteins immediately

(Gharagdaghi et al. 1999 Electrophoresis 20: 601-605)

____ Wash gel pieces 2 times in 50% HPLC grade methanol or ethanol for 10 min each.

____ Removed ALL of the excess liquid and store at –20oC.

Samples can be stored for months at –20oC.

____ If you are submitting more than 10 samples, please you a 96 well plate.

____ Label each tube or 96 well plate with YOUR NAME, DATE and SAMPLE NAME.

Location

The Johns Hopkins Mass Spectrometry and Proteomic Facility

725 N. Wolfe Street
Wood Basic Science Building, Room 504
Baltimore, MD 21205

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