Sample collection and sample amount

For bi-dimensional electrophoresis gels stained with colloidal Coomassie blue or with silver: the optimal gel thickness is one millimeter and the cut spots should be around 2 mm3.

For systematic analysis of proteins separated by mono-dimensional electrophoresis, the platform recommends to cut pieces in the central part of the lane (1-2 mm high, 1-2 mm wide).

Dispatch of the samples

For series containing less than 10 samples:

  • The cut gel pieces and shall be put in an 0.5 ml Eppendorf tube, ordered in annotated boxes with the name of the claimant.
  • The names of the spots must be indicated on the side and top of the tubes (with an indelible marker). Do not use a complex nomenclature; the optimal is to annotate tubes with increasing numbers.

For larger series (>10 samples):

  • The platform will send you specific 96-well plates. These plates are already drilled to be directly used by the digestion robot.
  • Keep the two first well empty. They will be used as controls to check the working of the automatic digestion.
  • Fill in the microplate table and send it by mail to your platform contact.

Silver stained gels

Gels can be silver stained but it is imperative that you do not use glutaraldehyde (protocole, french version only)

Spot discoloration will improve the process if it is realized quickly after staining (less than 24 h).

Several protocols of silver staining modified for mass spectrometry are available:

Contaminations

Althoug contaminations with keratins are not a major problem in LC-MS/MS (unlike with MALDI-TOF), several recommendations can be given to limit their presence and increase the analysis sensitivity.

  • Use freshly prepared buffer solutions, reserved for electrophoresis.
  • Use gloves (without powder and Teepol whased) during the whole experiment to avoid keratin and proteases traces on finger and gloves.

For liquid samples, salts should be in concentrations below 0,1M.

Detergent et signal suppression in ESI
Sodium dodecylsulfate (SDS)10 %
Sodium taurocholate10 %
Sodium cholate21-30 %
CTAB10 %
LDAO10 %
CHAPS31-60 %
Tween2010-20 %
Thesit10 %
Triton X10010 %
NP4010 %
n-octyl sucrose10-20 %
n-dodecyl sucrose10-20 %
n-dodecyl maltoside21-30 %
octyl glucoside21-30 %
octyl thioglucoside21-30 %
n-hexyl glucoside30-60 %
n-dodecyl glucoside60%

Ogorzalek Loo R.R.,. Protein Science 3: 1975 (1944)