小中大
Preparation for gel sample Precast gels (INVITROGEN, BioRad) Reduction and alkylation before gel run Reduce with DTT, DTE or TCEP Alkylate with Iodoacetamide See sample prep handbook Disolve the sample in SDS-buffer without reducing agent no -mercaptoethanol, no DTT, no DTE Reduce with 10 mM DTT, DTE, or TCEP Never use -mercaptoethanol !!!!!! Add 1/10 of the sample volume 100 mM stock solution DTT and DTE: 5 – 10 min at 100 C TCEP: 15 min at 60 C Cool down to RT Add 20 mM Iodacetamide and incubate for 30 min at RT Load and run gel Note: o Leave one lane free between each lane to be cut o Do not overload gel o Run the gel under optimal conditions (Voltage) Coomassie blue stain (colloidal preferred) If you use own Coomassie solution – make it fresh, also destainer Silver stained gels will be rejected Stain in new 10 x 10 cm quadratic containers Do not reuse old ones (contamination) Scan gel (pack between plastic sheets to avoid contamination) Clearly label lanes/bands/areas to be cut and analysed 2-D gel spots
