Abstract
BackgroundCell biologists face the need to rapidly analyse their proteins of interest in order to gain insight into their function. Often protein purification, cellular localisation and Western blot analyses can be multi-step processes, where protein is lost, activity is destroyed or effective antibodies have not yet been generated.AimTo develop a method that simplifies the critical protein analytical steps of the laboratory researcher, leading to easy, efficient and rapid protein purification, cellular localisation and quantification.ResultsWe have tagged the SMC2 subunit of the condensin complex with the Streptavidin-Binding Peptide (SBP), optimising and demonstrating the efficacious use of this tag for performing these protein analytical steps. Based on silver staining, and Western analysis, SBP delivered an outstanding specificity and purity of the condensin complex. We also developed a rapid and highly specific procedure to localise SBP-tagged proteins in cells in a single step procedure thus bypassing the need for using antibodies. Furthermore we have shown that the SBP tag can be used for isolating tagged proteins from chemically cross-linked cell populations for capturing DNA-protein interactions.ConclusionsThe small 38-amino acid synthetic SBP offers the potential to successfully perform all four critical analytical procedures as a single step and should have a general utility for the study of many proteins and protein complexes.
Highlights
Cell biologists face the need to rapidly analyse their proteins of interest in order to gain insight into their function
We have previously shown that Streptavidin-Binding Peptide (SBP)-tagged SMC2 protein of the condensin complex fully rescues function when stably expressed in conditional SMC2 knockout chicken DT40 cells [3]
SBP tag can be detected with a streptavidin fluorophore conjugate We investigated whether we could detect SBP-tagged proteins in a single step using the streptavidin-Alexa 488 fluorophore
Summary
Cell biologists face the need to rapidly analyse their proteins of interest in order to gain insight into their function. We have utilised the SBP tag to perform four separate cell biological and biochemical techniques on the SMC2 subunit of condensin, comparing the effectiveness of this technique using antibodies generated against SMC2. We have previously shown that SBP-tagged SMC2 protein of the condensin complex fully rescues function when stably expressed in conditional SMC2 knockout chicken DT40 cells [3]. There are two condensin complexes which share the same core SMC subunits (SMC2 and SMC4), but differ in their three auxiliary subunits (CAP-D2, H, G for condensin I and CAP-H2, D2, D3 for condensin II) Both condensin I and II are essential for proper chromosome organisation and packaging during mitosis and meiosis [4,5]
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