Immunoprecipitation protocol Experiment: Aim: Samples: 1 2 3 4 5 6 7 Proced re: 1!

"#a$ o t l%sates on ice 8 9 10 11 12 13 14

Date:

2! Add e& al amo nt o' proteins to eac# t (e! "otal protein ) ****** + Add l%sis ( ''er to t#e l%sates to ma,e t#e -ol me o' l%sates e& al! Sample .oncentration Amo nt needed /%sate 0ol /%sis ( ''er 0ol

3! Preclear l%sates! Add l%sates to (eads 1alread% $as#ed in 12P3S4 2times5! 6nc (ate at 4.7 308! 4! Add 0!541!0 + o' imm noprecipitatin+ anti(od% per t (e Anti(od%: Stoc, .oncentration: 0ol me added: 9inal .onc:

4! :otate: 4.7 2 #rs! Spin do$n l%sates at 4. to collect t#e sample! 5! ;as# 6+ (eads in 12 P3S 4 ******* l (eads are added to 1ml 12P3S7 "ap +entl% and in-ert 3 times! < ic, spin! Aspirate s pernatant! :epeat once more and store (eads on ice! 6! Add l%sates = Anti(od% mixt re to t#e (eads! "ap +entl% to s spend (eads! 7! :otate: >?@7 4.! 8! < ic, Spin7 aspirate s pernatant! 9! ;as# (eads: 3 times $it# 6P $as# ( ''er! 1 time $it# 12P3S=S0=S9! Spin do$n and aspirate s pernatant eac# time! 10! Add **** l 12 sample loadin+ ( ''er $it# 5A 34mer! "ap +entl%! 11! .ontrols: 4 4

12! 3oil: 100.7 5 min 13! Spin do$n and load s pernatant into +el! Gel Loading: 1! A o' +el and t#ic,ness sed: 2! @ m(er o' $ells: 3! Sample -ol me7 Bar,er -ol me: 4! 0olta+e r n7 "ime r n: 5! : n len+t#: 6! Sample order: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Transfer: 1! Bem(rane sed: 2! "rans'er n m(er C: 3! Amperes r n7 "ime r n: Ponceau: Stain 'or 28 $it# roc,in+7 destain $it# distilled $ater! :es lt: Blocking: :otate7 Blotting: 1! ;as# mem(rane 4 times $it# 12"3S"7 58 at :"!

2! Primar% anti(od%4 rotate7 Company: Isotype: Concentration: # Usage: 3! ;as# mem(rane 4 times $it# 12"3S"7 >nce $it# 12"3S7 58 at :"! 4! Secondar% anti(od% D rotate7 Company: Isotype: Concentration: # Usage: 5! ;as# mem(rane 4 times $it# 12"3S"7 >nce $it# 12"3S7 58 at :"! 6! Scan IR: IP as! "uffer composition #final concentrations in ater$ 50mB "ris pE 8!0 150mB @a.l 1mB Sodi m -anadate 1mB ED"A 1mB EF"A 0!1A "riton24100 %&PBS'Sodium (anadate'Sodium fluoride composition #final conc in %&PBS$ 1mB sodi m -anadate 10mB sodi m 'l oride