Purification of Human Multiprotein Complexes using OneSTrEP Technology (PROT41)

note 1 ). We recommend to isolate several clones and characterize them by a few simple criteria:

1. Western blotting to check expression level (see protocol) and proper cellular distribution of OneStrep-YPI (note 2 ). Antibodies against YPI should allow to determine the extent of overexpression as the tag introduces a mobility shift;
2. FACS analysis of DNA content to check the cell cycle profile;
3. Viability and cell growth (i.e. using Trypan blue and cell counting).

Protein extracts

We use a protocol for making cytosolic and nuclear extracts from HeLa S3 cells grown on dishes that has been developed in the Almouzni laboratory (modified from Li and Kelly (1984) and Martini et al. (1998)). These extracts are competent for in vitro DNA replication (Li and Kelly, 1984) and nucleosome assembly assays (Martini et al. 1998; Groth et al., 2005). In principal, any of your favourite lysis buffer could be used, but it is important to check that none of the components interfere with binding of the OneStrep-tag to the Strep-Tactin® matrix (IBA Tagnology website: http://www.iba-go.com/prottools/prot_fr01_01.html). We recommend the pre-fractionation step as it reduces the complexity of the starting material and can provide functional insights if the cytosolic and nuclear extract yield distinct complexes.

1. Grow cells on 150mm dishes until 80-90% confluence. We routinely use 6-8 150mm plates/experiment (note 3   and  note 4 ).
2. All following steps are done at 4°C in a cold room. Remove the medium and rinse cells 2x with ice-cold PBS. Drain well after the second wash.
3. Add 10 ml  Buffer E   (-inhib.)/150mm plate and incubate for 8-10 min to swell the cells.
4. Remove, drain and add 10ml of  Buffer E   (+inhib.)/150mm plate. Incubate for 8-10 min.
5. Remove and drain well by leaving the dishes in vertical position for about 2 min.
6. Scrape the cells and transfer into douncer (1 ml, Wheaton).
7. Homogenize using a loose pestle with 25 strokes.
8. Transfer to 2 ml Eppendorf tubes and pellet the nuclei by centrifugation at 1500 g for 5 min.
9. Transfer the supernatant to a new tube and clear it by spinning 15 min in a tabletop centrifuge at 4°C max. speed (13.000 rpm). This is your cytosolic extract containing cytoplasmatic and soluble nuclear proteins. Snap freeze and store at -80°C.
10. To the nuclei pellet from step 8 add one to two volumes of  Buffer N   and resuspend carefully with a P1000 pipette. Extract nuclear and chromatin bound proteins by end-over-end rotation 90 min at 4°C.
11. Spin 15 min in a tabletop centrifuge at 4°C max. speed. Transfer the supernatant to a new tube and snap freeze. This is your nuclear extract containing nuclear and chromatin bound proteins. (note 5 )
12. Wash the pellet (matrix and salt-extracted chromatin) once with  Buffer N , spin again, and snap freeze.

OneStrep-tag purification

We routinely use gravity flow  Strep-Tactin Superflow® columns   with a column volume (CV) of 200 µl (IBA Tagnology, Cat. No. 2-1207-550) (see  note 6 ). Purification is performed at 4°C in a cold room.

1. Equilibrate the column by applying 2x CV of  Washing buffer .
2. Add 5-10 mg of protein extracts to the column (not more than 1 ml).  note 7
3. Wash columns 10 times with 1 ml  Washing buffer .
4. Elute 6 times with 0,5 CV (100 µl)/elution step.  note 8   and  note 9
5. Snap freeze eluted fractions E1-E6. For the first test purification, collect also the flow-through and washes for the analyses described below.

Analysis of purified complexes

We quality check purified complexes by Western blotting (Figure 2 ) and silver staining (Figure 3 ). For Western blot detection we usually use 5-10 µl of E1-E6, and corresponding aliquots of input extracts, flow-through, and washes to judge the efficiency of binding and elution from the column. For silver staining we run 10-15 µl of E1-E6 to determine the purity. The purest fractions with highest yield can then be pooled and processed for mass spectrometry.

Western blot detection

1. Block membrane 1 hr at RT in 5% BSA, PBS-0.5%Tween.  note 10
2. Wash briefly 2x with PBS-0.1%Tween
3. Incubate membrane with Strep-tactin®-HRP (IBA Tagnology , Cat. No. 2-1502-001) 1:5000 diluted in blocking buffer at RT for 1 hr.
4. Wash 3x with PBS-0.1%Tween
5. Develop by chemiluminescence as usual (we use SuperSignal® West Pico Chemiluminescent Substrate, Pierce, Cat. No. 34080)

For detection of interacting proteins by mass spectrometry we precipitate proteins before separation of complexes on NuPAGE 4-12% Bis-Tris Gradient gels (Invitrogen, Cat. No. NP0321) and Coomassie staining (Figure 1 ). We have a good experience with methanol/chloroform precipitation protocol from Wessel and Flugge (1984).

Protein precipitation

1. To 200 µl of protein solution add 800 µl methanol.
2. Vortex and spin briefly.
3. Add 200 µl chloroform.
4. Vortex and spin briefly.
5. Add 600 µl H₂ O.
6. Vortex thoroughly for 30 seconds.
7. Centrifuge at 9000 g for 1 min.
8. The white precipitate will form the interphase. Remove the top phase.
9. Add 600 µl methanol
10. Vortex briefly.
11. Centrifuge at max. speed for 2 min.
12. Precipitate will end up at the bottom of tube. Remove the supernatant.
13. Speed vac until dry.
14. Resuspend in 1x SDS gel-loading buffer.

Back to top

Back to top