Materials and Reagents

Pipette tips (any brand)
100 mm cell culture dishes (IWAKI, catalog number: 4020-010 )
50 ml tubes (FALCON, catalog number: 352070 )
1.5 ml tubes (BIO-BIK, catalog number: RC-0150 )
Tris (hydroxymethyl) aminomethane (Tris.HCl) (NACALAI TESQUE, catalog number: 35409-45 )
Triton^TM X-100 (Sigma, catalog number: T8787 )
Phosphate buffered saline without Ca²⁺and Mg²⁺[PBS (-)] (NACALAI TESQUE, catalog number: 1148215 )
Protein assay dye reagent (Bio-Rad, catalog number: 500-0006 )
Sodium dodecyl sulfate (SDS) (Thermo Fisher Scientific, catalog number: BP166500 )
β-mercaptoethanol (Thermo Fisher Scientific, catalog number: BP176-100 )
HEPES (NACALAI TESQUE, catalog number: 17546-05 )
Sodium chloride (NaCl) (NACALAI TESQUE, catalog number: 31320-05 )
Glycerol (NACALAI TESQUE, catalog number: 17018-25 )
4-20% Mini-PROTEAN^® TGX^TM Precast Protein Gels (Bio-Rad, catalog number: 4561093 )
SilverQuest^TM silver staining kit (Thermo Fisher Scientific, catalog number: LC607 )
Protein markers (Bio-Rad, Precision Plus Protein^TM Standards-Dual Color, catalog number: 1610374 )
Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10270106 )
RPMI 1640 (Thermo Fisher Scientific, catalog number: 11875093 )
Benzonase^® Nuclease (Novagen, catalog number: 70746-3 )
cOmplete^TM, EDTA-free Protease Inhibitor Cocktail Tablets (Roche, catalog number: 4693132001 )
GFP-Trap^® Agarose (Chromotek, catalog number: gta-20 )
ANTI-FLAG^® M2 Agarose (Sigma, catalog number: A1205 )
3x FLAG^TM Peptides (Sigma, catalog number: F4799 )
Magnesium chloride hexahydrate (NACALAI TESQUE, catalog number: 7791-18-6 )
Dithiothreitol (DTT) (NACALAI TESQUE, catalog number: 14112-52 )
Bromophenol blue (Sigma-Aldrich, catalog number: B5525 )
Glycine (NACALAI TESQUE, catalog number: 17109-35 )
Methanol (Wako Pure Chemical Industries, catalog number: 136-09475 )
Quick CBB-stain (Wako Pure Chemical Industries, catalog number: 299-50101 )
Reticulocyte Standard Buffer (RSB) (see Recipes)
HEPES-Glycerol-NaCl (HGN165) (see Recipes)
Protease Inhibitor Cocktail (25x) (see Recipes)
Lysis Buffers 1 (LSB1) (see Recipes)
Lysis Buffer 2 (LSB2) (see Recipes)
Lysis Buffer 3 (LSB3) (see Recipes)
Lysis Buffer 4 (LSB4) (see Recipes)
Washing buffer (see Recipes)
FLAG^TM elution buffer (see Recipes)
2x-SDS sample buffer (see Recipes)
4x-SDS sample buffer (see Recipes)
10x-SDS-PAGE running buffer (see Recipes)
Semi-dry Western blot buffer (see Recipes)

Equipments

Pipettes (any brand)
Refrigerated swing-bucket centrifuge (Tomy, model: EX-136 )
Refrigerated fixed-angle centrifuge (Tomy, model: MX-301 )
Shaking incubator (TAITEC, model: BR-23FP )
Rotator (TAITEC, model: RT-50 )
Sonicator (Diagenode, model: Bioruptor^® Standard UCD-200)
Vortex mixer (Thermo Fisher Scientific, catalog number: 128101 )
Block heater (any brand)
Magnetic stand (Thermo Fisher Scientific; DynaMag^TM-2, model: 12321D )
Western blotting apparatus (Bio-Rad, model: Trans-Blot^® SD Semi-Dry Transfer Cell, catalog number: 1703940 )
Protein electrophoresis system (Bio-Rad, The Mini-PROTEAN^® Tetra cell, catalog number: 1658005EDU )
Imaging of Western blots (GE Healthcare, model: ImageQuant LAS 4000)

Procedure

Preparation of nuclear lysate
1. Collect 2 x 10⁷-3 x 10⁷ cells with culture medium in 50 ml conical tube and centrifuge at 800 x g for 3 min at 4 °C.
2. Discard the supernatant and suspend the cells in 10 ml of pre-chilled PBS and centrifuge at 800 x g for 3 min at 4 °C.
3. Discard the supernatant, suspend the cells in 1 ml of pre-chilled PBS, and transfer to 1.5 ml tube and centrifuge cells at 800 x g for 3 min at 4 °C.
4. Discard the supernatant, suspend the cells in 950 µl of pre-chilled RSB containing 1x protease inhibitor cocktail, and incubate at ice for 15 min.
5. Add 20 µl of 10% Triton^TM-X100 (v/v), invert tube 6-10 times, and centrifuge at 1,200 x g for 5 min at 4 °C.
6. Discard the supernatant (cytoplasmic fraction), suspend the nuclear pellet in 200 µl of pre-chilled LSB1 and mildly sonicate at Bioruptor^® for 5 min at low power (pulse 30 s, rest 60 s). Keep on ice for 5 min, and repeat sonication for 5 min at low power (pulse 30 s, rest 60 s). Rotate end-over-end for 1 h at 4 °C.
7. Centrifuge at 8,000 x g for 10 min at 4 °C and collect supernatant in fresh tube (S1).
8. Immediately suspend the pellet with the help of pipette tip in 150 µl of pre-chilled LSB2, and rotate end-over-end for 30 min at 4 °C.
9. Centrifuge at 8,000 x g for 10 min at 4 °C and collect supernatant in fresh tube. Add 50 µl of RSB containing 1x protease inhibitor cocktail to S2 and keep at ice (S2).
10. Immediately suspend the pellet with the help of pipette tip in 150 µl of pre-chilled LSB3, and rotate end-over-end for 30 min at 4 °C.
11. Centrifuge at 8,000 x g for 10 min at 4 °C and collect supernatant in fresh tube. Add 50 µl of RSB containing 1x protease inhibitor cocktail to S3 and keep at ice (S3).
12. Immediately suspend the pellet with the help of pipette tip in 150 µl of pre-chilled LSB4, and rotate end-over-end for 30 min at 4 °C.
13. Centrifuge at 8,000 x g for 10 min at 4 °C and collect supernatant in fresh tube. Add 100 µl of RSB containing 1x protease inhibitor cocktail to S4 and keep at ice (S4).
14. Pool extracts S1, S2, S3 and S4. Invert few times to mix and centrifuge at 3,000 x g for 5 min at 4 °C and collect supernatant in a fresh tube. Save 30 µl of pooled extract, add 30 µl of 2x SDS-sample buffer, and heat for 10 min at 95 °C. This will be used as input in western blotting.
Co-immunoprecipitation
1. Transfer 30 µl of GFP-Trap^® agarose for GFP-fused proteins or 30 µl FLAG^®-M2 agarose for FLAG^®-tagged proteins to 1.5 ml tube.
2. Add 500 µl of pre-chilled RSB, invert 4-6 times, and centrifuge at 800 x g for 3 min at 4 °C. Discard the supernatant carefully without disturbing the resin.
3. Transfer pooled nuclear lysates to the washed GFP-Trap^® agarose or FLAG^®-M2 agarose, and rotate end-over-end for 1-2 h at 4 °C.
4. Centrifuge at 80 x g for 3 min at 4 °C, and discard supernatant. Low speed centrifugation at this stage prevent precipitation of insoluble proteins aggregates that may have formed during incubation lysates with GFP-Trap^® or FLAG^®-M2 agarose at 4 °C. Discard the supernatant carefully without disturbing the resin.
5. To wash resin, add 800 µl of pre-chilled WB, invert 8-10 times, centrifuge at 800 x g for 3 min at 4 °C, and discard the supernatant carefully without disturbing the resin.
6. Repeat washing 3 more times, and discard the supernatants carefully without disturbing the resin. Proceed to elution of the bound proteins.
Elution
1. Elution by boiling
  1. Suspend resin in 100 µl 2x SDS-sample buffer, and heat for 10 min at 95 °C.
  2. Centrifuge at 1,800 x g for 3 min at 25 °C and collect supernatant in a fresh tube.
2. Elution by competition
  1. Suspend resin in 75 µl of 3x-FLAG^® elution buffer containing 500 µg/ml of 3x-FLAG^®peptides at 25 °C for 20 min with mild shaking.
  2. Centrifuge at 1,800 x g for 3 min at 25 °C and collect supernatant in a fresh tube.
  3. Add 25 µl of 4x SDS-sample buffer, and heat for 10 min at 95 °C.
Analysis of the immunoprecipitated proteins
1. Run 5-10 µl of input lysates and 10 µl of eluates (with SDS sample buffer) on SDS-PAGE gels followed by western blotting.
2. For silver staining, run 2-3 µl of input lysates and 10 µl of eluates (with SDS sample buffer) on SDS-PAGE gels and stain with SilverQuest^TM Silver Staining Kit.
3. For identification of proteins by mass spectrometry, eluates from 4-10 Co-IP experiments can be pooled. To load the pooled eluates in one single lane of SDS-PAGE, pooled eluates can be concentrated using a method that utilizes MS-compatible reagents such as methanol/chloroform based method. Run concentrated proteins on 4-20% SDS-PAGE gradient gel SDS-PAGE gels and stain with Quick-CBB stain. Cut the gel lane into 8-10 slices and process for MS analysis.

Notes

In a very first experiment, check the extraction of the protein that you want to immunoprecipitate in extracted fractions (S1 to S4) as well as insoluble pellet (Figure 1). If you do not see sufficient extraction, add additional extraction steps with higher Triton^TM X-100 and/or NaCl concentrations. Similarly, extractions with higher stringency buffer can be omitted if the protein is sufficiently extracted with low stringency buffers.
Pooled lysates prepared in this protocol can also be used for Co-IP using primary antibodies against the protein target of interest.
The pH of the each buffer was measured at room temperature.

Recipes

Reticulocyte Standard Buffer (RSB)
10 mM Tris-HCl, pH 7.4
5 mM MgCl₂
10 mM NaCl
HEPES-Glycerol-NaCl (HGN165)
10 mM HEPES-NaOH, pH 7.4
10% Glycerol (v/v)
165 mM NaCl
Protease Inhibitor Cocktail (25X)
Dissolve 1 tablet of cOmplete^TM, EDTA-free Protease Inhibitor Cocktail in 0.4 ml of water
Lysis Buffer 1 (LSB1)
HGN165 2 ml
Triton^TM X-100 (10% v/v) 20 µl
MgCl₂ (0.5 M) 8 µl
DTT (0.5 M) 4 µl
Benzonase^® (250 units/µl) 4 µl
Protease Inhibitor cocktail (25x) 130 µl
Lysis Buffer 2 (LSB2)
LSB1 500 µl
Triton^TM X-100 (10 % v/v) 10 µl
Lysis Buffer 3 (LSB3)
LSB1 500 µl
NaCl, 1 M 26.5 µl
Lysis Buffer 4 (LSB4)
LSB1 500 µl
NaCl, 1 M 53 µl
Wash buffer (WB)
HGN165 6 ml
Protease Inhibitor cocktail (25x) 240 µl
FLAG^TM elution buffer
50 mM Tris-HCl, pH 7.5
150 mM NaCl
0.1% Triton^TM X-100 (v/v)
2x-SDS sample buffer
120 mM Tris-HCl, pH 6.8
20% glycerol (v/v)
2% SDS (w/v)
0.04% bromophenol blue
10% β-mercaptoethanol (v/v)
4x-SDS sample buffer
120 mM Tris-HCl, pH 6.8
40% glycerol (v/v)
4% SDS (w/v)
0.08% bromophenol blue
20% β-mercaptoethanol (v/v)
10x-SDS-PAGE running buffer
Tris base 30.3 g
Glycine 188 g
SDS 10 g
Water to 1 liter
Semi-dry Western blot buffer
Tris base 6 g
Glycine 14.4 g
SDS 1 g
Methanol 200 ml
Water to 1 liter

Acknowledgments

We are grateful for the financial supports from the Japan Society for the Promotion of Science (KAKENHI 15H05784), and a Collaborate Research Grant with Ono Pharmaceutical Co., Ltd. to T.H., and a Grant-in-Aid for Scientific Research (C) 16K07214 from the Japan Society for the Promotion of Science (to N.A.B.). The IP protocol is adapted from Husain et al. (2016).

Competing interests

The authors declare no competing financial interests.

References

Husain, A., Begum, N. A., Taniguchi, T., Taniguchi, H., Kobayashi, M. and Honjo, T. (2016). Chromatin remodeller SMARCA4 recruits topoisomerase 1 and suppresses transcription-associated genomic instability. Nat Commun 7: 10549.
Kim, N. and Jinks-Robertson, S. (2012). Transcription as a source of genome instability. Nat Rev Genet 13(3): 204-214.
Kobayashi, M., Aida, M., Nagaoka, H., Begum, N. A., Kitawaki, Y., Nakata, M., Stanlie, A., Doi, T., Kato, L., Okazaki, I. M., Shinkura, R., Muramatsu, M., Kinoshita, K. and Honjo, T. (2009). AID-induced decrease in topoisomerase 1 induces DNA structural alteration and DNA cleavage for class switch recombination. Proc Natl Acad Sci U S A 106(52): 22375-22380.
Kobayashi, M., Sabouri, Z., Sabouri, S., Kitawaki, Y., Pommier, Y., Abe, T., Kiyonari, H. and Honjo, T. (2011). Decrease in topoisomerase I is responsible for activation-induced cytidine deaminase(AID)-dependent somatic hypermutation. Proc Natl Acad Sci U S A 108(48): 19305-19310.
Pommier, Y. (2006). Topoisomerase I inhibitors: camptothecins and beyond. Nat Rev Cancer 6(10): 789-802.
Smith, A. L., Friedman, D. B., Yu, H., Carnahan, R. H. and Reynolds, A. B. (2011). ReCLIP(reversible cross-link immuno-precipitation): an efficient method for interrogation of labile protein complexes. PLoS ONE 6: e16206.