|Home | About | Journals | Submit | Contact Us | Français|
Tandem affinity purification (TAP) is a generic two-step affinity purification protocol for isolation of TAP-tagged proteins together with associated proteins. We used bacterial artificial chromosome to heterologously express TAP-tagged murine Sgo1 protein in human HeLa cells. This allowed us to test the functionality of the Sgo1-TAP protein by RNA interference-mediated depletion of the endogenous human Sgo1. Here, we present an optimized protocol for purification of TAP-tagged Sgo1 protein as well as KIAA1387 from HeLa cells with detailed instructions. The purification protocol can be completed in 1 day and it should be applicable to other proteins.
Most cellular processes are carried out by multiprotein complexes. The identification of individual subunits is essential for understanding their function. To streamline the purification of protein complexes from cells, the TAP protocol was developed1,2. It has been successfully applied to purify protein complexes from various organisms including bacteria, yeasts, plants as well as mammalian cells3-14. We used the TAP protocol to purify proteins associated with murine Sgo1 protein15.
It is important to confirm that tag addition does not significantly affect the function of the tagged protein. In model organisms such as yeast, functionality of tagged proteins can be easily tested. In mammalian cells, the lack of efficient homologous recombination makes the functionality test difficult. Kittler et al.16 demonstrated that expression of murine bacterial artificial chromosomes (BAC) in human cells provides a reliable method to create RNA interference (RNAi)-resistant tagged transgenes. In such cells, the endogenous human gene can be knocked down by RNAi, while the corresponding murine gene expressed from the integrated BAC resists the RNAi treatment. We expressed murine Sgo1-TAP at physiological levels in HeLa cells from a BAC integrated into the HeLa genome15. This allowed us to determine whether the murine Sgo1-TAP was able to complement functionally the phenotype caused by depletion of the endogenous human Sgo1. As a control we used HeLa cells expressing untagged murine Sgo1 from a BAC integrated into the HeLa genome. Depletion of Sgo1 in HeLa cells by RNAi leads to precocious separation of sister chromatids17,18. In contrast, in RNAi-treated cells expressing the murine SGO1-TAP transgene, the levels of precocious sister chromatid separation dropped considerably toward wild-type levels (Fig. 1). These results demonstrate that the murine Sgo1-TAP is functional in HeLa cells as the SGO1-TAP transgene prevented precocious separation of sister chromatids caused by depleting endogenous human Sgo1 by RNAi. Notably, the rescue of the RNAi phenotype of SGO1 by BAC transgenesis also confirms the specificity of the RNAi experiment17. In addition to HeLa cells described here, we have succeeded in generating BAC-transgenic U2OS cells with the same protocol (data not shown). Given the fact that other BAC-transgenic cell lines, including mouse ES cells, have been successfully generated, it is likely that BAC-transgenic clones can also be generated from many different cells. However, it is probable that for other cell lines, different transfection protocols have to be used for the delivery of the BAC DNA.
As an example of possible applications for this system, we used murine Sgo1-TAP expressed from a BAC integrated into the HeLa genome to identify proteins that interact with Sgo1 in mitotic cells. Murine Sgo1-TAP, but not a control protein KIAA1387-TAP, associated with a specific form of protein phosphatase 2A (Tables (Tables11 and and2).2). This finding helped us to elucidate the mechanism by which Sgo1 protects centromeric cohesion15,19. Notably, endogenous human Sgo1 also associated with murine Sgo1-TAP, suggesting that Sgo1 proteins form homo-oligomers (Table 1). Thus, our protocol also allows detection of oligomer formation of the tagged protein.
Here, we describe an optimized TAP purification protocol with detailed instructions. We also describe our mass spectrometry (MS) protocol (Step 20A) and our SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining protocol to visualize purified proteins (Step 20B; Fig. 2), which we have developed specifically for our target protein but could potentially be adapted for other proteins.
Prepare standard DMEM medium supplemented with 10% fetal calf serum, 0.2 mM l-glutamine, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin.
50 mM Tris pH 8.0, 150 mM NaCl, 10% glycerol (v/v), 0.2% NP-40 (v/v), Complete Mini Protease Inhibitor (Roche) (1 tablet per 10 ml) and 1 mM PMSF. ▲ CRITICAL Add inhibitors just before use.
50 mM Tris pH 8.0, 150 mM NaCl, 10% glycerol (v/v) and 0.1% NP-40 (v/v).
50 mM Tris pH 8.0, 150 mM NaCl, 10% glycerol (v/v), 0.1% NP-40 (v/v), Complete Mini Protease Inhibitor (Roche) (1 tablet per 10 ml) and 1 mM PMSF. ▲ CRITICAL Add inhibitors just before use.
10 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol (v/v), 0.1% NP-40 (v/v), 0.5 mM EDTA and 1 mM DTT. ▲ CRITICAL Add DTT just before use.
10 mM β-mercaptoethanol, 10 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol (v/v), 0.1% NP-40 (v/v), 1 mM imidazole, 1 mM Mg-acetate and 2 mM CaCl2.
1 mM β-mercaptoethanol, 10 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM Mg-acetate and 2 mM CaCl2.
50% methanol (v/v), 12% acetic acid (v/v) and 0.018% formaldehyde (v/v).
0.1% silver nitrate (w/v) and 0.027% formaldehyde (v/v).
6% (w/v) sodium carbonate, 0.00002% Na2S2O3 (w/v) and 0.018% formaldehyde (v/v).
The UltiMate system consists of an UltiMate μHPLC pump and a UV detection unit with the nano UV-Z View flow cell with 3 nl cell volume, the Switchos μ-column-switching device with loading pump and two 10-port valves, and the FAMOS μ-autosampler equipped with the 250 μl sample loop, 15 μl injection needle and 250 μl syringe (Large Volume Injection Kit). For FAMOS, use the μL-Pickup injection method. As a transport liquid on FAMOS use 0.1% aqueous TFA (in Reagent Transport Vial 1). Use the 300 μm ID × 5 mm length trap column cartridges for sample trapping and cleaning before separation. Operate the trap column at 20 μl min−1 on Switchos loading pump. As a separation column, use the 75 μm ID × 150 mm length nano separation column. Operate the separation column at 275 nl min−1 on UltiMate separation system. Use the following tubing for separation: to deliver the flow from Switchos to the FAMOS autosampler and to transport the sample to the trap column, use 130 μm ID tubing. Keep the tubing length as short as possible. To connect the trap column to the Switchos valve, use the provided cartridge holder and two 30 μm ID fused silica connections, which are delivered with the trap column. To make all flow paths used for nano flow (275 nl min−1), use the 20 μm ID PEEKSil connection tubing. To connect the UV cell output with the MS inlet, use 20 μm ID fused silica and keep it as short as possible. Make all fluidic connections from Ultimate nano HPLC to the mass spectrometer using 20 μm ID fused silica capillaries, connected with low dead-volume micro tight connectors from Dionex.
Use the following mobile phases for the separation column:
For separation, use the following HPLC gradient:
|Time (min)||% B|
Use the following tubing for separation: to deliver the flow from Switchos to the FAMOS autosampler and to transport the sample to the trap column, use 130 μm ID tubing. Keep the tubing length as short as possible. To connect the trap column to the Switchos valve, use the provided cartridge holder and two 30 μm ID PEEKSil connections delivered with the trap column. To make all flow paths used for nano flow (275 nl min−1), use the 20 μm ID PEEKSil connection tubing. To connect the UV cell output with the MS inlet, use 20 μm ID fused silica and keep it as short as possible.
Transfer the eluting peptides online to a heated capillary of an ion trap mass spectrometer. Use the following ESI parameters:
|Spray voltage||1.5 kV|
|Capillary temperature||200 °C|
|Capillary voltage||26 V|
|Tube lens offset voltage||95 V|
|Electron multiplier||−800 V|
The collision energy is set automatically depending on the mass of the parent ion.
■ PAUSE POINT Cell pellets can be frozen in liquid nitrogen and stored at −80 °C for several days.
■ PAUSE POINT Calmodulin beads can be frozen in liquid nitrogen and stored at −80 °C for several days.
▲ CRITICAL STEP While the conventional TAP protocol uses EGTA to elute proteins from calmodulin beads, we were not able to efficiently elute proteins from calmodulin beads by EGTA. To overcome this limitation, we directly submitted calmodulin beads to tryptic digestion followed by MS analysis and we boiled calmodulin beads in SDS buffer for the SDS-PAGE and silver staining analysis.
The protein complex purification protocol (Steps 2–20) can be completed in 1 day.
Step 1: growing HeLa cells, about 3 days
Steps 2 and 3: harvesting HeLa cells, 2 h
Steps 4–20: protein complex purification, 11 h
Step 20A(i)–(vi): tryptic digest, 1.5 days
Step 20A(vii) and (viii): nano HPLC and MS, 5 h
Step 20A(ix): database search, 4 h
Step 20B: SDS-PAGE and silver staining, 3 h
Troubleshooting advice can be found in Table 3.
We developed this TAP protocol to optimize purification of proteins associated with mammalian Sgo115. Although each protein has unique properties, we believe that our TAP protocol should be applicable to other proteins. However, the functionality test of TAP-tagged murine transgenes is limited to those where human homologs can be identified. In some cases, artifactual binding partners may be identified owing to differences between human and mouse proteins. Although the conventional TAP tag has proven to be a very useful tool for protein complex purification, alternative affinity binding moieties are now available that may provide higher protein complex yield or allow for both protein isolation and live imaging of protein localization6,23.
This work was supported by Boehringer Ingelheim and Austrian Science Fund (P18955-B03). Work in the Mechtler lab was supported by the Austrian Proteomics Platform (APP) within the Austrian Genome Program (GEN-AU) and by the Mitocheck project within the Sixth Framework Program of the European Commission. J.G. was a recipient of the EMBO long-term fellowship. We thank Tony Hyman for the gift of the TAP tagging cassette, and Laurence Pelletier and Ralf Kittler for help with Sgo1 tagging. We also thank members of Karl Mechtler lab for help with MS analysis and Jan Michael Peters and members of his lab for helpful discussions.
COMPETING INTERESTS STATEMENT The authors declare no competing financial interests.
Rights and permissions information is available online at http://npg.nature.com/reprintsandpermissions