Nuclear/Cytoplasmic Fractionation of Proteins from Caenorhabditis elegans

Nuclea /Cy oplasmic F ac iona ion o P o eins om Caeno habdi is elegans Alejand o Ma a-Cabana1, Olga Sin2,3, Renée I. Seins a4, and Ellen A. A. Nollen4,* 1Depa men o Gene ics, Uni e si y o Se ille, Se ille, Spain 2Max Planck Resea ch G oup o RNA Biology, Max Planck Ins i u e o Molecula Biomedicine, Müns e , Ge many 3Cells-in-Mo ion Clus e o Excellence, Uni e si y o Müns e , Müns e , Ge many 4Eu opean Resea ch Ins i u e o he Biology o Ageing, Uni e si y o G oningen, Uni e si y Medical Cen e G oningen, G oningen, he Ne he lands Abs ac C. elegans is widely used o in es iga e biological p ocesses ela ed o heal h and disease. To s udy p o ein localiza ion, luo escen ly- agged p o eins can be used in i o o immunohis ochemis y can be pe o med in whole wo ms. He e, we desc ibe a echnique o localize a p o ein o in e es a a subcellula le el in C. elegans lysa es, which can gi e insigh in o he loca ion, unc ion and/o oxici y o p o eins. Keywo ds C. elegans ; Subcellula ac iona ion; P o ein localiza ion; Nucleus; Cy oplasm; Immunoblo Backg ound Subcellula ac iona ion has been used in di e en model o ganisms o iden i y and s udy p o ein unc ion in nuclei, memb anes and cy oplasm. Fo example, agg ega ion-p one p o eins may be mo e oxic when hey a e localized in he nucleus o in he cy osol (Kon opoulos e al ., 2006; Ba mada e al ., 2010). He e we p o ide a p o ocol (adap ed om Chen e al., 2000 and La Rocca e al ., 2007) o localize speci ic p o eins in he nuclea and cy oplasmic ac ions o C. elegans . Ma e ials and Reagen s 1. 94 mm pla es (G eine Bio One In e na ional, ca alog numbe : 633185) 2. 15 ml conical ube (SARSTEDT, ca alog numbe : 62.554.502) 3. 1.5 ml ubes (G eine Bio One In e na ional, ca alog numbe : 616201) exclusi e licensee Bio-p o ocol LLC. *Fo co espondence: [email p o ec ed]. Compe ing in e es s The au ho s decla e no con lic s o in e es o compe ing in e es s. Eu ope PMC Funde s G oup Au ho Manusc ip Bio P o oc. Au ho manusc ip ; a ailable in PMC 2018 No embe 20. Published in inal edi ed o m as: Bio P o oc . ; 8(20): . doi:10.21769/BioP o oc.3053. Eu ope PMC Funde s Au ho Manusc ip s Eu ope PMC Funde s Au ho Manusc ip s 4. Pelle pes le (Sigma-Ald ich, ca alog numbe : Z359947-100EA) 5. Glass slides (Fishe Scien i ic, ca alog numbe : 12164682) 6. Ni ocellulose (Bio-Rad Labo a o ies, ca alog numbe : 1620112) 7. C. elegans s ain 8. Esche ichia coli OP50 s ain 9. Deionized wa e (dH2O) 10. Choles e ol (Fishe Scien i ic, ca alog numbe : 10263660) 11. E hanol absolu e (Me ck, ca alog numbe : 1009831000) 12. Magnesium sulpha e (MgSO4) (Fishe Scien i ic, ca alog numbe : 10264630) 13. Calcium dichlo ide (CaCl2) (Fishe Scien i ic, ca alog numbe : 10171800) 14. di-Po assium hyd ogen phospha e (K2HPO4) (Me ck, ca alog numbe : 105101) 15. Po assium dihyd ogen phospha e (KH2PO4) (Me ck, ca alog numbe : 104873) 16. Casein diges , Di co (BD, ca alog numbe : 211610) 17. Selec aga (The mo Fishe Scien i ic, In i ogen™, ca alog numbe : 30391049) 18. Disodium hyd ogen phospha e (Na2HPO4) (Ac os O ganics, ca alog numbe : 424380010) 19. Sodium chlo ide (NaCl) (Me ck, ca alog numbe : 106404) 20. DL-Di hio h ei ol (DTT) (Sigma-Ald ich, ca alog numbe : D0632) 21. HEPES (Sigma-Ald ich, ca alog numbe : H4034) 22. Po assium hyd oxide (KOH) (Fishe , ca alog numbe : 10705921) 23. Po assium chlo ide (KCl) (Fishe Scien i ic, ca alog numbe : 10010310) 24. Magnesium dichlo ide (MgCl2) (Fishe Scien i ic, ca alog numbe : 10518060) 25. E hylenediamine e aace ic acid (EDTA) (Sigma-Ald ich, ca alog numbe : E6758) 26. Suc ose (Fishe Scien i ic, ca alog numbe : 10386100) 27. Tween 20 (Sigma-Ald ich, ca alog numbe : P1379-100ML) 28. P o ease inhibi o s (cOmple e) (Roche, ca alog numbe : 11697498001) 29. Benzonase nuclease (Me ck, ca alog numbe : 70746) 30. Pie ce™ BCA P o ein Assay Ki (The mo Fishe Scien i ic, ca alog numbe : 23225) 31. Liquid ni ogen 32. Ammonium pe sul a e (APS) (The mo Fishe Scien i ic, ca alog numbe : A/ 6120/60) Ma a-Cabana e al. Page 2 Bio P o oc . Au ho manusc ip ; a ailable in PMC 2018 No embe 20. Eu ope PMC Funde s Au ho Manusc ip s Eu ope PMC Funde s Au ho Manusc ip s 33. N , N , N ′, N ′-Te ame hyle hylenediamine (TEMED) (Fishe Scien i ic, ca alog numbe : 10142863) 34. 40% Ac ylamide/Bis solu ion (29:1) (Bio-Rad Labo a o ies, ca alog numbe : 1610146) 35. Sodium dodecyl sul a e (SDS) (Me ck, Calbiochem, ca alog numbe : 428015) 36. T is Base (Roche Diagnos ics, ca alog numbe : 11814273001) 37. Hyd ochlo ic acid 37% (Ac os O ganics, ca alog numbe : 124630010) 38. Glycine (Fishe Scien i ic, ca alog numbe : 10070150) 39. Me hanol (Me ck, ca alog numbe : 106009) 40. B omophenol blue (Ac os O ganics, ca alog numbe : 403160100) 41. Glyce ol (Sigma-Ald ich, ca alog numbe : G5516) 42. β-Me cap oe hanol (Me ck, ca alog numbe : 444203) 43. Milk powde (Campina) 44. PageRule ™ Plus P es ained P o ein Ladde (The mo Fishe Scien i ic, ca alog numbe : 26619) 45. Ame sham ECL P ime Wes e n Blo ing De ec ion Reagen (GE Heal hca e, ca alog numbe : RPN2236) 46. An ibodies (see Table 1) 47. M9 bu e (see Recipes) 48. Phospha e bu e ed saline (PBS) (see Recipes) 49. PBS-T (see Recipes) 50. Blocking solu ion (see Recipes) 51. Nema ode g ow h medium (NGM) pla es (see Recipes) 52. Phospha e bu e (see Recipes) 53. Hypo onic bu e (see Recipes) 54. Hype onic bu e (see Recipes) 55. 25x p o ease inhibi o s (see Recipes) 56. 1 M DTT (see Recipes) 57. 12% ac ylamide gel (see Recipes) 58. Running bu e (see Recipes) 59. T ans e bu e (see Recipes) 60. SDS lysis bu e (see Recipes) Ma a-Cabana e al. Page 3 Bio P o oc . Au ho manusc ip ; a ailable in PMC 2018 No embe 20. Eu ope PMC Funde s Au ho Manusc ip s Eu ope PMC Funde s Au ho Manusc ip s Equipmen 1. Single channel pipe es (Gilson, models: P2G, P20G, P200G, P1000G) 2. O bi al shake (The mo Fishe Scien i ic, model: MaxQ™ 2000) 3. 20 °C incuba o (Winecoole , LIEBHERR, model: WK 4126) 4. S e eo mic oscope (Leica, model: MZ7.5) 5. Pelle pes le (mo o ) (Sigma-Ald ich, ca alog numbe : Z359971) 6. Table op cen i uge, cooled (Eppendo , model: 5424 R) 7. Cen i uge (The mo Fishe Scien i ic, model: SL 40R) 8. Au ocla e (VWR, model: VAPOUR-Line Li e) 9. -80 °C eeze (Sanyo, model: VIP plus) 10. Mini PROTEAN 3 sys em (Bio-Rad Labo a o ies, ca alog numbe : 1658001edu) 11. WB Image (GE Heal hca e, model: ImageQuan LAS 4000 mini, ca alog numbe : 28955813) So wa e 1. ImageJ (Open sou ce: h ps://imagej.nih.go /ij/) 2. Mic oso Excel (Mic oso Co po a ion, Redmond, USA) P ocedu e A. Collec ion o wo ms 1. P epa e 10-15 pla es o bleach synch onized wo ms, wi h app oxima ely 800-1,000 wo ms pe 9 cm NGM pla e in o de o ge ela i ely la ge nuclea ac ions. No e: We pe o med subcellula ac iona ion expe imen s wi h as ew as 5 x 9 cm NGM pla es pe condi ion. Howe e , wo king wi h 10 x 9 cm NGM pla es will yield a mo e wo kable nuclea ac ion, as he pelle will be mo e easily isible du ing he washing s eps . 2. Incuba e wo ms a 20 °C o app oxima ely 72 h, un il day 1 o adul hood (D1). No e: Subcellula ac iona ion can be pe o med a di e en ages o wo ms. He e, we desc ibe a p o ocol o wo ms a he i s day o adul hood (D1). Howe e , he p o ocol is also easible o L4-s aged animals and olde wo ms. One should keep in mind ha i is easie o b eak he cu icle o la ae wi h a pelle pes le han olde wo ms. Thus, he numbe o s okes should be adjus ed acco dingly o he age o he wo ms (7-12 imes) . Ma a-Cabana e al. Page 4 Bio P o oc . Au ho manusc ip ; a ailable in PMC 2018 No embe 20. Eu ope PMC Funde s Au ho Manusc ip s Eu ope PMC Funde s Au ho Manusc ip s 3. Collec D1-s aged wo ms wi h ~4 ml M9 pe pla e in a 15 ml conical ube and wash un il he supe na an is clea (3-5 imes). 4. Wash he wo m pelle wice wi h 1 ml cold hypo onic bu e . 5. P epa e 2-5 ml ‘comple e hypo onic bu e ’ pe sample by adding 1 M DTT ( inal concen a ion: 1 mM DTT) and 25x p o ease inhibi o s ( inal concen a ion: 2x p o ease inhibi o s) o a ew ml o s anda d hypo onic bu e . No e: I is sugges ed o calcula e he olume o ‘comple e hypo onic bu e ’ ha is equi ed o he expe imen . Pipe e he equi ed olume o hypo onic bu e in a ube and add DTT and p o ease inhibi o s; compounds ha should bo h be s o ed a -20 °C . 6. Remo e all hypo onic bu e and add ‘comple e hypo onic bu e ’, as p epa ed in S ep A5 (~500 μl). T ans e he wo m suspension in o a 1.5 ml ube. No e: Es ima e he olume o he wo m pelle and add he same olume o ‘comple e hypo onic bu e ’ . 7. A oid eezing he wo m pelle a his poin , as his may damage he nuclei and cause leakage o nuclea p o eins in he cy osol. B. F ac iona ion 1. Homogenize wo ms wi h a pelle pes le o 1 min and le he wo ms es o 1 min, epea his 5 o 10 imes. Keep he wo ms on ice du ing he whole p ocess. Pipe e 5 μl o he wo m suspension on o a glass slide a e 4-5 imes g inding he pelle wi h he pes le o e alua e he deg ee o wo m lysis. Homogeniza ion is comple e when 70%-80% o he wo ms a e dis up ed and only a ew wo m emnan s a e isible (see Figu e 1). 2. Pelle he wo m bodies and deb is by spinning a 500 x g , 4 °C o 5 min. 3. T ans e he supe na an o a new 1.5 ml ube and spin again a 500 x g , 4 °C o 5 min o make su e you ge id o all he wo m deb is. 4. T ans e he supe na an o a new 1.5 ml ube. Sa e 25-30 μl o his ac ion in a new 1.5 ml ube (‘inpu ac ion’) and keep on ice. W i e down he o al olume o supe na an . Disca d he pelle . 5. Pelle he nuclei a 4,000 x g , 4 °C o 5 min. 6. T ans e he supe na an o a new 1.5 ml ube and cen i uge a maximal speed (17,000 x g ), 4 °C o 30 min. T ans e he supe na an o a new 1.5 ml ube; his will con ain he ‘cy oplasmic ac ion’. Ma a-Cabana e al. Page 5 Bio P o oc . Au ho manusc ip ; a ailable in PMC 2018 No embe 20. Eu ope PMC Funde s Au ho Manusc ip s Eu ope PMC Funde s Au ho Manusc ip s 7. Wash he pelle om S ep B5 wi h 500 μl ‘comple e hypo onic bu e ’ (as p epa ed in S ep A5) and cen i uge he samples a 4,000 x g , 4 °C o 5 min. 8. Disca d he supe na an and add 500 μl o esh ‘comple e hypo onic bu e ’ (as p epa ed in S ep A5) o esuspend he nuclea pelle and pipe e he suspension in o a new 1.5 ml ube. Cen i uge he samples a 4,000 x g , 4 °C o 5 min. No e: I is impo an o change ubes a e each wash, as ubulin and a y subs ances end o s ick o he ube walls, which may con amina e he nuclea ac ion . 9. P epa e ‘comple e hype onic bu e ’ by adding 1 M DTT ( inal concen a ion: 1 mM DTT) and 25x p o ease inhibi o s ( inal concen a ion: 2x p o ease inhibi o ) o he hype onic bu e . 10. Remo e he supe na an and dissol e he pelle in a small olume o ‘comple e hype onic bu e ’ (as p epa ed in S ep B9) (hal o o less he olume ha was no ed in S ep B4). W i e down he olume ha was used o dissol e he nuclea ac ion. T ans e he suspension o a new 1.5 ml ube; his will con ain he ‘nuclea ac ion’. 11. OPTIONAL: Fo be e p o ein ex ac ion add 25 U/μl o Benzonase nuclease o he ‘nuclea ac ion’ and incuba e he sample a 4 °C o 45 min on an o bi al shake . 12. De e mine a his poin he p o ein concen a ion o he ‘inpu ’ (~20 μg/ μl), ‘cy osolic ac ion’ (~18 μg/μl) and ‘nuclea ac ion’ (~5 μg/μl) wi h he Pie ce™ BCA P o ein Assay Ki . 13. Sa e s opping poin : Flash eeze samples in liquid ni ogen and s o e a -80 °C un il u he use (see Figu e 2 o a schema ic o e iew o he ac iona ion s eps). C. Wes e n blo ing 1. Thaw he samples con aining he ‘inpu ’, ‘cy osolic ac ion’ and ‘nuclea ac ion’. Pipe e a olume o he samples (ei he he same amoun o p o ein o all ac ions o he same olume) in o new 1.5 ml ubes. No e: When equal olumes a e loaded, one can ac ually compa e he amoun o speci ic p o ein be ween he di e en ac ions wi hin a sample. When equal p o ein amoun s a e loaded, one can look o an en ichmen o a p o ein wi hin speci ic ac ions be ween condi ions o wo m s ains. Impo an ly, always w i e down he olume o he o al inpu ac ion and he olume in which he nuclea ac ion is dissol ed. En iching he nuclea ac ion, i.e., dissol ing he pelle in less olume, inc eases he concen a ion o p o eins, which may be use ul du ing immunoblo ing. Howe e , in his si ua ion he signal canno be Ma a-Cabana e al. Page 6 Bio P o oc . Au ho manusc ip ; a ailable in PMC 2018 No embe 20. Eu ope PMC Funde s Au ho Manusc ip s Eu ope PMC Funde s Au ho Manusc ip s compa ed di ec ly o ha o he inpu , as his ac ion is no en iched in he same way. The e o e, co ec ions based on he desc ibed olumes should be pe o med a e wa d . 2. Add ¼ olume o SDS lysis bu e (5x). 3. Boil he samples a 95 °C o 10 min. 4. P epa e a 12% SDS polyac ylamide gel. 5. Load he samples and he p o ein ma ke on o he 12% SDS polyac ylamide gel. Fill he ank wi h unning bu e and s a un a 100 V un il he samples ha e un h ough he s acking gel and hen con inue a 120-140 V un il he dye on eaches he bo om o he gel. 6. Blo he gel o a ni ocellulose memb ane wi h a we ans e in he ans e bu e a 95 V o 55-60 min a 4 °C. 7. Block he ni ocellulose memb ane o 1 h in blocking solu ion. OPTIONAL: Cu he ni ocellulose memb ane jus below he 70 kDa ma ke (LMN-1 no mally uns a ound 70 kDa and ubulin a ound 55 kDa). Bu keep in mind he size o you p o ein o in e es . 8. Incuba e he memb ane wi h he app op ia e an ibodies o 1 h a oom empe a u e o o e nigh a 4 °C (see Table 1). LMN-1 is used as a nuclea ma ke (~70 kDa) and ubulin (~55 kDa) as a cy osolic ma ke . 9. Wash he memb anes 3 x 10 min wi h 1x PBS-T. 10. Incuba e he memb anes wi h he app op ia e HRP-conjuga ed seconda y an ibodies o 1 h a oom empe a u e (see Table 1). 11. Wash he memb anes 3 x 10 min wi h 1x PBS-T. 12. Add he ECL o he memb anes and de elop he blo s wi h he Las 4000 mini. OPTIONAL: When no cu ing he memb anes, one can s ip he memb anes and e-p obe wi h a di e en an ibody o in e es . Da a analysis Figu e 3 shows an example o an immunoblo . A leas h ee independen expe imen s should be pe o med and an example o da a analysis can be ound in Figu es 5 and S5C o Sin e al. (2017)s. The ac ion le els we e quan i ied by densi ome y using ImageJ. A de ailed u o ial is a ailable a h ps://imagej.nih.go /ij/docs/menus/analyze.h ml (unde “Gels Submenu”) and a h p://lukemille .o g/index.php/2010/11/analyzing-gels-and- wes e n-blo s-wi h-image-j/. To calcula e he cy osol/inpu a io, he signal o in e es can be no malized agains he signal o he cy osolic ma ke (α- ubulin). The nuclea ac ion is a concen a ed sample, and Ma a-Cabana e al. Page 7 Bio P o oc . Au ho manusc ip ; a ailable in PMC 2018 No embe 20. Eu ope PMC Funde s Au ho Manusc ip s Eu ope PMC Funde s Au ho Manusc ip s he e o e one should use he inpu olume and he olume in which he nuclea ac ion was esuspended o calcula e he “ eal” alue. Recipes 1. M9 bu e (1 L) KH2PO43 g Na2HPO46 g NaCl 5 g dH2O 1,000 ml Au ocla e a 121 °C o 20 min 1 M MgSO41 ml (add a e au ocla ing) 2. PBS (1 L) NaCl 8 g KCl 0.2 g KH2PO40.24 g Na2HPO41.44 g dH2O 1,000 ml HCl Adjus pH o 7.2-7.6 Au ocla e a 121 °C o 20 min 3. PBS-T PBS con aining 0.01% Tween 20 4. Blocking solu ion 5% Milk in PBS-T 5. NGM pla es (1 L) NaCl 3 g Selec Aga 17.5 g Casein diges 7.5 g Au ocla e a 121 °C o 20 min and hen cool o ~55 °C A e cooling add he ollowing bu e s: Ma a-Cabana e al. Page 8 Bio P o oc . Au ho manusc ip ; a ailable in PMC 2018 No embe 20. Eu ope PMC Funde s Au ho Manusc ip s Eu ope PMC Funde s Au ho Manusc ip s 1 M MgSO41 ml 1 M CaCl21 ml Choles e ol (5 mg/ml in e hanol) 1 ml 1 M Phospha e bu e 25 ml 6. Phospha e bu e 1 M pH 6.0 (1 L) KH2PO4118 g K2HPO423 g Au ocla e a 121 °C o 20 min 7. Hypo onic bu e HEPES KOH pH 7.6 15 mM KCl 10 mM MgCl25 mM EDTA 0.1 mM Suc ose 350 mM 8. Hype onic bu e HEPES KOH pH 7.6 15 mM KCl 400 mM MgCl25 mM EDTA 0.1 mM Tween 20 0.1% Glyce ol 10% 9. 25x p o ease inhibi o s Dissol e 1 able Comple e in 2 ml dH2O (s o e a -20 °C) 10. 1 M DTT Dissol e 0.154 g DTT in 1 ml dH2O (s o e a -20 °C) 11. 12% ac ylamide gel (1.0 mm casse e) Running Gel S acking Gel dH2O 2.2 ml 1.74 ml Ma a-Cabana e al. Page 9 Bio P o oc . Au ho manusc ip ; a ailable in PMC 2018 No embe 20. Eu ope PMC Funde s Au ho Manusc ip s Eu ope PMC Funde s Au ho Manusc ip s