SILAC LABELLING

(23.11.10 Maria)

Reagents

Medium and serum

- DMEM-10 (lacking Arg and Lys); Dundee Cell Prod.; cat. no. LM010

- Dialysed FBS (dFBS); Dundee Cell Prod.; cat. no. DS1003

Light amino acids

- L-Arg0, SIGMA; cat. no. A-8094

- L-Lys0; SIGMA; cat. no. L-8662

- L-Pro0; SIGMA; cat. no. P-5607

Heavy amino acids

Two options in the UK: Sigma and CK Gas

SIGMA:

- L-Arg6; cat. no. 643440-SPEC

- L-Arg10; cat. no. 608033-SPEC

- L-Lys4; cat. no. 616192-SPEC

- L-Lys8; cat. no. 608041-SPEC

CK Gas:

- L-Arg6; cat. no. CLM-2265-H-1

- L-Arg10; cat. no. CNLM-539-H-1

- L-Lys4; cat. no. DLM-2640-1

- L-Lys8; cat. no. CNLM-291-H-1

Check on availability and price before ordering.

Conditions

Light: DMEM-10 containing 10% dFBS, 84 mg/L Arg0, 146 mg/L Lys0 and 200 mg/L Pro0

Medium: DMEM-10 containing 10% dFBS, 84 mg/L Arg6, 146 mg/L Lys4 and 200 mg/L Pro0

Heavy: DMEM-10 containing 10% dFBS, 84 mg/L Arg10, 146 mg/L Lys8 and 200 mg/L Pro0

When comparing 2 conditions, light (Arg0, Lys0) and heavy (Arg10, Lys8) labelling are used.

Procedure to label cells

Split cells 5-8 passages with a dilution of 1:5 or higher using the different media. In my hands, cells were 96-98% labeled after five passages and 98% after seven passages.

Check labelling by running samples on SDS/PAGE, in-gel digestion and MS. MS raw files can be analysed with Proteome Discoverer or MaxQuant to quantify labeling.

Labelled cells can be kept in liquid N₂ for future use.