Stable Isotope Labeling with Amino acids in Cell culture (SILAC) Kits From Invitrogen

Nowadays, mass spectrometry (MS) techniques have developed into remarkably useful tools for proteomics research, specifically for protein identification and modification. However, if your goal is to quantitatively differentiate protein expression levels between control and experimental groups, then MS does have some obstacles. A big improvement for quantitative proteomics research was achieved with the development of the SILAC (Stable Isotope Labeling with Amino acids in Cell culture) technique and as a result, it has become a very popular method for that.

Invitrogen provides two different types of SILAC kits depending upon what type of protein you are extracting and which type of cell culture media you will use. The SILAC Phosphoprotein ID and Quantitation Kit provides a special lysis buffer which contains a lot of different phosphatase inhibitors. Invitrogen provides the composition of this lysis buffer, but does not provide specific inhibitor concentrations. To identify the expression of membrane-bound proteins, such as receptors, there is the SILAC Membrane Protein ID and Quantitation Kit which contains a membrane lysis buffer. This buffer makes it much easier to get membrane proteins which are normally are very hard to extract. Each kit is available with either RPMI-1640 media or DMEM. We used the SILAC Phosphoprotein Identification and Quantitation Kit (cat#SP10005) with RPMI-1640 media. Each SILAC kit provides everything you need for the experiment (protocol, fetal bovine serum, media, and labeled amino acid) except antibiotics. The SILAC RPMI-1640 or DMEM media is lysine- and arginine-free so additional lysisne and arginine should be added before the experiment; one of these amino acids can be used as your label, such as [¹³C₆]-Lys (experimental group) and [¹²C₆]-Lys (control group). We used this kit with Jurkat cells grown in RPMI media with [¹³C₆]-Lys (experimental group) and [¹²C₆]-Lys (control group). We generally use a standard lysis buffer such as RIPA for our experiments.

SILAC is very simple and straightforward. Two populations of cells (control group and experimental group) are grown in cell culture media such as RPMI-1640 or DMEM. However, one of the cell populations is grown with normal amino acids, the other is grown with labeled amino acids. For example, the experimental group is grown with lysine labeled with ¹³C (heavy), [¹³C₆]-Lys, and the control group is grown with the normal lysine, ¹²C (light), [¹²C₆]-Lys. When the cells are growing in each labeled media, the lysine (heavy or light) is incorportaed into all of proteins in the cells. Therefore, all of the peptides in the experimental group which contain lysine are 6 Da (per lys) heavier than the control group. Because only newly synthesized proteins will contain the labeled amino acid, a number of cell divisions in the labeled media are required for this experiment. After six rounds of cell division in labeled media in our experiment, 100% of cells contain the labeled lysine. Several different stable isotopes, such as deuterium, ¹³C, ¹⁵N could be used for labeling. For the MS analysis, the same number of cells from the experimental group ([¹³C₆]-Lys) and control group ([¹²C₆]-Lys) were combined after counting the cells with an hemocytometer. Combined cells were lysed in the same tube and proteins were loaded onto SDS-PAGE and then visualized by comassie staining. This is the reason that we could rely on our SILAC experiments: lysis of cells from both groups in the same tube and then subsequent running on the same SDS-PAGE minimized the error between control and experimental groups. The several bands which were indicated as interesting targets were cut out and digested by trypsin or another appropriate protease. The digested peptides were analyzed by MS. The reduced chance of error due to pipetting/handling during sample preparation gave us increased confidence in our results and is an important advantage for quantitative proteomics research.