ࡱ> 695] <$bjbj .&!b!b Rxx 2228j$$2t()++++++$6O-O|111)1)1110=101l l 1l 11OOl xB : Yeast Protein Isolation All reagents should be at 4(C and samples handled on ice until 2min spin Spin down 1-1.5x107 log-phase cells in a 2ml tube, wash cells with 1 ml water You can freeze the cell pellet in N2 and process later This can be scaled up 5x increase the volume of all the reagents by 5x except the acetone, do two washes with 1ml of acetone* *for low cell number process samples straight after MEP Rapid Aging Purification, include 1x cOmplete Protease Inhibitors in buffers during MEP purification Re-suspend cell pellet in 100(l water Add 15(l 2N NaOH w/ 80mM DTT (freshly added), vortex, ice for 10 minutes Add 15(l 50% TCA, vortex, ice for 10 minutes Samples can be held indefinitely at 4( at this point Spin 2min 14,000rpm at RT Pipette off supernatant into TCA waste, add 1ml ice-cold acetone Invert to mix then spin 2min 14,000rpm at RT Pipette off acetone and dry briefly For normal samples Re-suspend pellet in 40(l sample buffer, vortex and heat at 65( for 5 min Vortex again until pellet is gone (pipetting can help too) Heat 5 min at 65( again For low cell number Resuspend in 20(l of Laemlli buffer containing freshly added 100mM DTT Heat 5 mins at 95C Spin the sample for 30s at top speed and transfer supernatant to new tube, store -20( Quantification and loading: Measure concentration by OD280. Use buffer including DTT as a reference since DTT absorbs at this wavelength Add 10x Orange G to each sample to 1x final concentration before loading For samples in Sample buffer: Heat 10min at 65(, spin briefly and load on gel (Dont place samples on ice as they will solidify!) For samples in Laemlli buffer: Heat 5min at 95(, spin briefly and load on gel Sample buffer: 10ml: 70mM Tris pH6.8 700l of 1M 8M urea 4.8g 5% SDS 2.5ml 20% (the good stuff from Biorad) 1mM EDTA 20l of 0.5M water to 9ml, warm gently to dissolve 100mM DTT Add from 1M stock just before use Laemlli buffer: 60mM Tris pH 6.8 10% Glycerol 2% SDS (from the high quality 20% stock) Make this to 90% final volume, then just before use add 10% 1M DTT (giving final concentration of 100mM DTT) 10x Orange G: 0.1% Orange G in water  7.4% -mercaptoethanol can be used, but the process needs to be performed in the fume hood  don t heat samples above 70( in this buffer as the proteins will aggregate  Diluting and measuring by Bradford assay is probably more accurate      FILENAME 45acmuvh i j l      & ' 6 7 B E F L M ] e f g ļܑjhl0JUhUoh>ThT[t jmhYhhV hV >*hV hV hV hii h'{H*h'{h=hKrj h0H*h0hl jh>-h>-hYhYh{5CJ(\aJ(hYhY5CJ(\aJ(2bcj k  / 0 z { ; < j k gdYgdV ^gdiigd'{gdKrj$a$gdY * 3 < I j k : ; B C D V W Y g h ٿٵٿٱ٪ٱٟٱٱь jhV!hiih/OJQJhD jmh/ hh/h/jh0JU jhh!= jmhYhV!hehh>Th([hlh=h>-hYhT[t jhT[t7 * B C X ) * 3ghgdV gd/gdii`gdgdgdY ) H I 123fgh/0Fļ~zrzhlOJQJhljhl0JU hVEhV!hehii hVEhiihiimH sH hehemH sH heh!=mH sH h>-mH sH hmH sH  jhhV jhV!h/hjh0JUhV!hV!hV!6ha,0JvOFgdgdY ^`gdiigdV FH$$$$$'$+$,$-$.$/$5$6$7$8$9$:$;$<$ټ hVEhV!jhP0JU*hD0JmHnHu* hP0JjhP0JUhdBh"hZGLmHnHuUhPjhPUjhIJUhIJhjh0JU ,$-$9$:$;$<$gdY Yeast Protein Isolation v1.7 Houseley lab  PAGE 2 ,1h. A!"#$% s2 0@P`p2( 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p8XV~ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@_HmH nH sH tH @`@ NormalCJ_HaJmH sH tH DA`D Default Paragraph FontRiR  Table Normal4 l4a (k (No List 4@4 PHeader  9r 4 @4 PFooter  9r .)@. P Page Number>@"> ii Footnote TextCJaJ<1< iiFootnote Text Char@&`A@ iiFootnote ReferenceH*PK![Content_Types].xmlN0EH-J@%ǎǢ|ș$زULTB l,3;rØJB+$G]7O٭Vc:E3v@P~Ds |w< LHJ ]J -& DDQQQT F<$  <$  /ELNT!8@0(  B S  ?<@Xanuw~(.     H K iHIOO1fg   9 ; < F K iHIOO1fgK 32-UV p#}sVE>-0!=@/GgJ}JZGLPjS>T([gKrjUoT[t{'{S~eZ!o/Y_DN[7t dBlKV!ii=al8}>5 IJ" @(  J h@hhH@UnknownG*Cx Times New Roman5Symbol3.*Cx ArialC.,{ @Calibri Light7.@CalibriA$BCambria Math"1hL eGR| KXXq4  2 HP ? 2!xx΂d^ Yeast Protein IsolationHouseley Jon HouseleyOh+'0  8 D P \hpxYeast Protein Isolation HouseleyNormalJon Houseley11Microsoft Office Word@5z @~M@›@=X՜.+,0 hp  Edinburgh Univeristy  Yeast Protein Isolation Title  !"#$&'()*+,./0123478;Root Entry F =:@1Tablel WordDocument .&SummaryInformation(%DocumentSummaryInformation8-MsoDataStore@=0=SR0RUYI0==2@=0=Item 2PropertiesUCompObj r   F Microsoft Word 97-2003 Document MSWordDocWord.Document.89q