While we believe the purifications presented to be among the highest quality preparations of the human exosome and NEXT complexes produced by a single step affinity isolation at the given scale, the expected coprecipitation patterns of specific interactors have previously been established using affinity isolation and protein staining (17, 31). However, to our knowledge, Figure 4 also presents the first example of a Coomassie-stained coprecipitation pattern for the human CBC, and this result is well supported by published results obtained using other analytical means: the interaction between NCBP1 and NCBP2 is well-established and leads to formation of a heterodimer that binds to m7G-capped RNA (32); KPNA2 (human importin-α) is required for the nuclear export of CBC bound to capped RNAs and the subsequent re-import of CBC along with importin-β (44, 45); PHAX was shown to be a bridging factor between CBC and CRM1, an export factor for U small nuclear RNAs (snRNA)(46); and SRRT (ARS2) was reported to interact with the CBC and shown to be crucial for microRNA (miRNA) biogenesis (47). While it is not certain how many different complexes this purification comprises—or in what proportions these other proteins interact with the CBC in concert or separately—it will be interesting to determine unambiguously if the PHAX containing CBC population overlaps with the SRRT-containing population, as these two components have not previously been reported in mutual coprecipitation, and they are considered to have differing physiological roles in RNA metabolism.
Polyclonal Ig versus nanobody
Conducting identical purifications of RBM7-LAP using magnetic beads conjugated with either affinity isolated anti-GFP polyclonal Ig or a bacterially expressed and purified anti-GFP nanobody (28, 29), we observe the loss of the nonspecific Fc binding protein TRIM21 (data not shown) from our isolations and, as expected, reduced pollution from Ig chains when the nanobody is used. However, at equivalent proportions by mass used in coupling to Dynabeads, the anti-GFP nanobody reagent exhibited lower yields than our polyclonal Ig (Supplementary Figure S5).
Here we have applied an optimized workflow for the isolation of endogenous protein complexes from human cells at quantities detectable by Coomassie staining of SDS-PAGE gels, with low nonspecific background, and without protein over-expression. Expression at or near the endogenous level mitigates the possibility of induced artifacts that can lead to erroneous conclusions. Using magnetic beads coupled to high-affinity antibodies, 100 mg WCW of material typically affords superior quality isolations for most protein complexes we have examined, even at low stringency of extraction—providing yields in the range of tens to hundreds of nanograms per protein band, amenable to identification by MALDI-TOF MS. We often use 50 mg WCW, corresponding to 1/2 of one 150-cm2 culture dish, to great success in pilot experiments.
It is often desirable to make several grams of cell grindate at one time and to store it at -80°C for a subsequent period of continued experimentation. (We have done so 6 months or more with no apparent loss of performance in affinity isolations.) Most of the time we have achieved excellent initial results with the usual “standard buffers” (e.g., HEPES, pH 7.4, 100 mM NaCl, 0.5% v/v Triton X-100). However, as individual experiments do not consume exorbitant quantities of material, it is reasonable to explore several affinity isolation conditions in parallel, rapidly converging on an optimized result with efficient use of time and material. A main advantage of 3×FLAG-based and GFP-based affinity isolations is the ability to use high stringency conditions. Therefore, we commonly screen increasing NaCl concentrations up to 1 M, as well as numerous different detergents at different concentrations (e.g., Triton X-100, Tween-20, CHAPS).
Once a favored result is identified, it is simple to scale-up several-fold if required for downstream analysis. Due to the yield and purity that can be achieved, native release of protein complexes via competitive elution, or protease cleavage of the tagged protein, provide the possibility of carrying out enzymatic or biochemical assays, as well as biophysical analyses such as negative stain EM.
This work was funded by the Lundbeck and Danish National Research Foundations as well as National Institutes of Health (NIH)/National Center for Research Resources’ (NCRR) grant no. 5 U54 RR022220-06. Dr. Ina Poser and Prof. Anthony Hyman provided the RBM7-LAP cell line, and Prof. Andrzej Dziembowski provided the bacterially expressed and purified GFP nanotrap reagent. We thank Drs. Samson Obado and Michela Di Virgilio for assistance with experimental procedures and critical reading of the manuscript. This paper is subject to NIH Public Access Policy.
The authors declare no competing interests.
Correspondence Address correspondence to John LaCava, Laboratory of Cellular and Structural Biology, The Rockefeller University, 1230 York Avenue, New York, NY, USA. Email: [email protected]">[email protected]References
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