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InterPlay Mammalian TAP System - Details & Specifications

The InterPlay Mammalian TAP System allows you to recover interacting proteins from mammalian cells. The novel method is based on expression of a protein of interest fused to two affinity tags: a streptavidin binding peptide (SBP) and a calmodulin binding peptide (CBP). Tandem affinity purification yields your tagged protein and interacting proteins using gentle washing and small molecule elution conditions. You do not need protease digestion to recover interacting protein partners since SBP and CBP elute easily from their affinity resins, leaving your interacting proteins in-tact.

Two-Step Purification Protocol

Purifying proteins with the TAP protocol requires two steps (Figure 1). Since each purification step has a different sort of background, the combined protocol yields exceptionally clean proteins. For the first purification step, apply the mammalian cell lysate to a streptavidin column; then elute using biotin. Apply that eluate to a calmodulin column for the second purification step, and release the protein of interest and its binding partners by removing calcium from the affinity resin with a chelator such as EGTA. Because elution conditions are so gentle, the interacting proteins are not disrupted, and you may further analyze the proteins using techniques like Western blotting and mass spectrometry.

Figure 1: To purify with the TAP protocol, apply the mammalian cell lysate to the streptavidin resin, then elute using biotin, and apply that eluate to a calmodulin resin.

Proven Purification of Interacting Proteins

To demonstrate that we can purify known interacting partners, we co-transfected Cos7 cells with myocyte-enhancing factor 2A (MEF 2A) tagged at the N-terminus with SBP and CBP and myocyte-enhancing factor 2C (MEF 2C) with an N-terminal FLAG tag. We grew and harvested cultured cells, and then purified the proteins using the TAP protocol. To characterize the TAP-purified proteins, we resolved the protein complex by SDS-PAGE, stained with Coomassie Brilliant Blue staining stock solution (Figure 2a and 2b), and isolated and purified the proteins from the gel. Using in-gel trypsin digestion followed by MALDI mass spectrometry analysis, we identified MEF 2A and 2C, indicating that the TAP-tagged MEF 2A protein interacted and co-purified with its partner, MEF 2C.

Innovative Vector Design

The pNTAP and pCTAP vectors allow you to clone your gene of interest with the SBP and CBP tags fused to either the N- or C-terminus of your gene. The vectors take advantage of the high-level constitutive cytomegalovirus (CMV) promoter and the SV40 poly(A) tail. Expression of the neo/kanr cassette is under control of the SV40 promoter. Both the N- and C-terminal vectors are supplied in all three reading frames (see vector maps).

Complete System

The InterPlay Mammalian TAP systems consist of all of the vectors, resins, and buffers you need to conduct a TAP experiment. The two systems offer either N- or C-terminal fusion of your protein of interest to the CBP and SBP tags, and each vector is supplied in all three reading frames.

For Research Use Only. Not for use in diagnostic procedures