30% Acrylamide 7.88 ml ml ml. Tris (ph 8.8) 4.5 ml 6.5 ml 9 ml. APS 135 ul 195 ul 270 ul. TEMED 6.75 ul 9.75 ul 13.

Transcription

1 WESTERN BLOT Kyle Nvakwski Nvember 9 th, 2012 Bwdish Lab, McMaster University Hamiltn, ON, Canada PROTOCOL Pre-wrk: Turn heat blck t 100C (If making lysates) Allw prtease inhibitr (Hpper, upper shelf) t defrst at RT (If making lysates) Mnday: Seed Tuesday: Transfect Thursday: Cllect & Start Western Making gels: 1. Clean an apprpriate number f 1mm glass plates & frnt cver plates. Assemble the plates int the green hlder. Place a grey rubber piece n the white 2-gel hlder and add the plates. Test the seal by adding water between the glass plates and wait 2-5 minutes. Dump water and dry with a kimwipe if seal is k. 2. Fr a single 10ml 12% separating gel, mix: 2.3ml H ml 30% acrylamide mix 2.0ml 1.5M Tris (ph 8.8) (w/ SDS) 60uL 10% APS (Bwdish chemical cabinet; lasts 1 week at -20C) Needs t be made fresh; 100mg APS in 1mL H 2 O. 3uL TEMED Ntes: Add APS and TEMED immediately befre puring the gel int the mld Can make extra mix as a means t check fr gel plymerizatin Can be dne the day befre if required. Stre at 4 C. 2 Gels 3 Gels 4 Gels H 2 O 5.18 ml 7.48 ml ml 30% Acrylamide 7.88 ml ml ml Tris (ph 8.8) 4.5 ml 6.5 ml 9 ml APS 135 ul 195 ul 270 ul TEMED 6.75 ul 9.75 ul 13.5 ul 3. Pur gel int mld and tp up with 70% EtOH. Wait ~40min fr gel t plymerize.

2 I suck up 1mL int a blue pipette tip and let it rest n the bench. When it is nt pssible t expel the liquid (ie it has plymerized) frm the tip, the gel is set. 4. After plymerizatin, pur ut EtOH and wash with MilliQ water. 5. Fr a single 5ml stacking gel, mix: Fr a single gel: 1.3ml H20 320uL 30% acrylamide mix 234uL 1.0M Tris (ph 6.8) (w/ SDS) 20uL 10% APS 1.5uL TEMED Ntes: Add APS and TEMED immediately befre puring the gel int the mld Can make extra mix as a means t check fr gel plymerizatin 2 Gels 3 Gels 4 Gels H 2 O 2.93 ml 4.26 ml 5.85 ml 30% Acrylamide 720 ul 1040 ul 1.44 ml Tris (ph 6.8) 530 ul 761 ul 1053 ul APS 45 ul 65 ul 90 ul TEMED 3.38 ul 4.86 ul 6.75 ul 6. Pur gel int mld and insert cmb. Wait ~10min fr gel t plymerize. 7. After plymerizatin, remve cmb and wash with MilliQ water t remve bubbles. Preparing samples: 1. Aspirate media ff cells. Wash with 2.5ml PBS. Remve PBS. 2. Mix 25l prtease inhibitr per 5ml f NP40 (r RIPA) lysis buffer. 3. Add 250l lysis buffer slutin t each well. 4. Incubate plates at -80 C fr 10min. Grab ice bucket. 5. Scrape all cells int slutin. 6. Transfer slutin t Eppendrf tubes and spin at 4C and 13,000rpm fr 10min. Discard pellet. Keep supernatants n ice. Can freeze at -80 C and stp at this pint 8. Add 250l DTT per 750l 3 sample buffer. 9. Add 50l 3 sample buffer t 100l cell slutin. Extra 900l f samples can be stred at -80 C. 10. Bil sample fr 10 min. Can freeze at -80 C and stp at this pint

3 Running and transferring samples: 1. Assemble the MiniPROTEAN cassette accrding t manufacturer s instructins. Use dam n far end if there is an dd number f samples 2. Pur 1.2L 1 running buffer int the MiniPROTEAN cassette tank. Fill tank t tp mark 5x stck, make t 1x by diluting 240mLs f 5x stck t 1.2L with MilliQ water. 3. Lad maximum 30l f samples int the wells in the MiniPROTEAN cassette. Use 7.5l prtein ladder where necessary (Precisin Plus Kalidescpe Cmmn Reagents -20 C) Use lng tips t lad samples 4. Run fr 40min at 200V and RT. Can use 110V fr 75 minutes. 20 minutes; the gel may require lnger. Run until the dye is rughly at the green part near the bttm f the MiniPROTEAN cassette. 5. Make transfer buffer (1x) 100mL 10x transfer buffer, 100mL methanl, 800mL MilliQ water. OPTIONAL: Sak the gel in transfer buffer fr 15 min t 1 hur. 6. Sak 2 pieces f Whatman paper (per gel) in transfer buffer alng with the spnges (RIGHT BEFORE) Assemble in transfer buffer inside the pyrex dish 7. Sak PVDF membrane in methanl until wet (in little bx) Mark PVDF membranes t differentiate them use PEN Needs t be cut t crrect size (use sizer piece f paper) Remember t remve the wells (stacking gel prtin) frm the gel using a green cutter. 8. Assemble sandwich : Black plastic -> Spnge -> Whatman paper -> Gel -> PVDF membrane (upside dwn) -> Whatman paper -> Spnge -> White plastic Make sure there are n bubbles between the gel and PVDF membrane; rll with a 15 ml tube. 9. Place sandwich in MiniPROTEAN cassette. Pur 1L transfer buffer int tank. Easy way t remember: Runs t the light, s the black part f the sandwich shuld face the black part f the cassette, the prtein will travel twards the light side f the sandwich (int the PVDF). Add small ice pack t tank 10. Run fr 70min at 70V and 4C. Antibdy staining: 1. Incubate PVDF membrane rcking in 20ml blcking slutin fr 1hr at RT (r 4C vernight). 2. Add 1 Ab t blcking slutin. Incubate 1hr at RT (r 4C vernight). 3. Discard blcking slutin. Wash PVDF membrane 3 with 10ml TBST fr 10min per wash. Just eyeball it, make sure its fully cvered. Can leave fr 1hur if needed 4. Add 2 Ab t a fresh prtin f blcking slutin. Incubate rcking fr 30min at RT. MAX 1 HOUR. Fr anti-muse and anti-rabbit use at 1:100,000 t 1:200,000 (if using the nes frm Jacksn that we have). Fina has a wrking slutin f anti-muse (Franklin, Fina s shelf). Use at 1:10,000 dilutin (2l per 20ml). Anti-rat (ED31) wrking slutin is available in the HRP bx (Curie, tp shelf). Use at 1:20,000 dilutin (1l per 20ml)

4 5. Discard blcking slutin. Wash the PVDF membrane 3 with 10ml TBST fr 10min per wash. Visualizing samples: 1. Place a piece f Parafilm n a flat surface and place the membrane prtein side up n tp. If mre than 1 membrane, verlap arund 0.5cm. 2. Mix tgether slutins in the ECL kit accrding t the manufacturer s instructins. Use ~2ml per gel. 1mL f each slutin ECL Kit is GE Healthcare White/Orange bx in Fridge DO NOT MIX TWO SOLUTIONS UNTIL RIGHT BEFORE (ie when gel is n parafilm) 3. Place membrane in cassette and expse t film. Start with 2min and expse fr lnger perids afterwards as necessary. Fld film in the tp right crner Dn t swish it arund, just place prperly the first time Antibdy stripping (ptinal): 1. Incubate membrane in MeOH fr 10min. 2. Remve MeOH. Wash 3 with TBST fr 5min per wash. 3. Apply 20ml stripping buffer. Incubate rcking fr 1hr. D this twice, changing buffer in between. 4. Remve stripping buffer. Wash 3 with TBST fr 5min per wash. 5. Incubate membrane in 8ml f blcking slutin fr 1hr. 6. Apply 1 Ab. Incubate vernight. 7. Cntinue at Step 3 f Antibdy staining.

5 Prtcls fr making Western bltting slutins: Running buffer Made frm a 5 stck. Add 960ml MilliQ H 2 O t 240ml stck slutin (5x). Making 5 stck: 15.1g Tris base (25mM) 94g glycine (250mM, ph = 8.3) Add 500ml MilliQ H 2 O 50ml 10% SDS (0.1%) Tp up t 1L with MilliQ H 2 O. Transfer buffer Made frm a 10 stck. Add 800ml MilliQ H 2 O and 100ml MeOH t 100ml stck slutin. Making 10 stck: Blcking slutin 5% skim milk pwder disslved in TBST 100ml slutin = 5g milk pwder + 100ml TBST Stir fr 20min Making 1L TBST: In large flasks by cmputer 100ml 10 TBS 900ml MilliQ H 2 O 1ml Tween 20 Stir 5min Making 10 TBS: 500mM ph 7.4 Tris 1500mM NaCl