Cell surface proteins represent a key subset of the cell's total protein, most notably because of the high concentration of integral membrane proteins. These proteins play major roles in signal transduction, cell adhesion, ion transport and serve as common pharmacological targets. Specific labeling of surface proteins is a useful tool to analyze this subpopulation of cellular proteins. Implementing biotin labels is one approach to achive this goal.
The Pierce® Cell surface Protein Isolation Kit from Thermo Scientific contains all the necessary reagents for optimal biotinylation and the subsequent isolation of mammalian cell surface protein from eight samples consisting of four 90-95% confluent T75 flasks, including all the wash-buffers needed. Very sensitive reagents like the Dithiothreitol (DTT) and the EZ-Link Sulfo-NHS-SS-Biotin are provided in eight separately packed microtubes or vials, respectively. The entire kit can be stored at 4ºC.
In our laboratory, this kit is used on a regular basis to label cell surface proteins on platelets under different experimental conditions. After the labeled protein is isolated, the protein mixture gets separated using two dimensional gel electrophoresis.
The major advantage of this kit is its straightforward protocol, which is very detailed, but easy to follow. In addition, the kit enables an easy biotin labeling using the membrane-impermeable EZ-Link Sulfo-NHS-SS-Biotin, ensuring specificity of the labeling reaction. Since the biotinylation reagent is thiol-cleavable, the bound proteins can easily be released using a SDS-PAGE sample buffer containing 50 mM DTT.
The first step of the protocol is the labeling reaction using the reconstituted EZ-Link Sulfo-NHS-SS-Biotin. The cells get incubated with the biotin for 30 minutes at 4ºC. In my experiments, the cells are sensitive to cold conditions, therefore, I did the labeling step at room temperature for only 15 minutes and it worked just fine. After the incubation period, 500 ul of the quenching solution are added to every 10 ml of labeling reaction to stop the labeling process. The cells get harvested in a pellet and subsequently lysed using the included lysis buffer. For this step, additional protease inhibitors are required which are not included in the kit. Next, the labeled proteins get isolated using the NeutrAvidin Agarose which needs to be gently swirled to get an even suspension, which is indeed very important for consistent results. The provided columns get loaded with the agarose, washed and then loaded with the cell lysate. The columns get incubated at room temperature for 60 minutes, best on an end-over-end rotator. In my experience, the MacsMix from Mitenyi worked very well for this application. After adding additional protease inhibitors to the provided wash buffer, the column gets washed and finally the protein of interest gets eluted. The elution is based on the cleavage of the EZ-Link Sulfo-NHS-SS-Biotin by DTT, which gets added to a SDS-PAGE sample buffer which again is not part of the kit. The eluted protein can be used for Western blot analysis. In our case, we cleaned the sample from its elution buffer to resuspend it in a 2D compatible buffer.
For us, the kit worked consistently in a reproducible way, which made it a useful tool in protein expression analysis.
Research Assistant Professor
Department of Surgery, Division of Vascular Surgery
University of Utah