ࡱ> RUQ[ DXbjbj .Ziiii&PF F 8OLL (xzzzzzz$!{$9DDDxDxDDDpZ&yDd0 D%p%D%D DR %F Y : Western blotting This protocol is designed for empty Invitrogen Novex gel cassettes and Novex blotting unit Pouring and running the gel Stand the empty gel cassette on a paper towel somewhere it cant easily fall over (for example, leaning against the side of a plastic box) Using the ratios given in Sambrook and Russell appendix 8, mix water, Tris, acrylamide and SDS to make 10ml resolving gel per gel cassette. * *for low cell number, when working the aging time-course, use a 15% acrylamide (30% solution, 37.5:1, Biorad) gel To a 10ml aliquot of resolving gel, add the necessary amount of 10% APS, invert to mix, then add TEMED1 and mix by inversion again Pour ~8ml into the gel cassette it should reach the second plastic ridge Layer 500(l water on top of the gel add this down the side of the cassette slowly Once the gel has set (about 30min), draw off the water layer with a tissue To 2.5ml of 5% stacking gel, add 25l 10% APS, invert to mix, then 2.5l TEMED, invert to mix again Pipette the stacking gel into the top of the gel cassette (being careful to avoid bubbles), and carefully insert the comb (some acrylamide will dribble out, catch this with paper towel) Once set (10min), remove the comb and wash the gel and comb under running water Denature protein samples in loading dye for 3 min at 95( or 10 min at 65 (if using high urea loading dye), and defrost BioRad ladder Meanwhile, mount the gel in the tank and pour in 1x Tris-glycine running buffer (~700ml). Make sure the buffer isnt leaking from upper to lower chamber Cool the protein samples to room temperature, spin briefly and load into wells using a Biorad extended tip. Load 5l ladder. Run at 75V until samples reach the resolving gel then increase to 175V As the ladder is prestained, it is relatively easy to judge how far to run the gel Blotting* Make 500ml transfer buffer and soak the blotting pads in ~400ml Cut 1 sheet of PVDF membrane per gel to 8x7cm and wet for 15s in methanol, then soak for 5min in transfer buffer. Cut two sheets of Whatman 3MM per gel to same size, wet these with transfer buffer just before use Open the gel cassette, cut off the wells and the foot of the gel, DO NOT RINSE THE GEL Put 1 sheet of wetted 3MM paper on the gel, then use the gel knife to remove from cassette onto parafilm so the gel is face up. Lay the membrane carefully on the gel, followed by the other sheet of wetted 3MM paper. Roll over a pipette to remove bubbles Repeat the above steps for a second gel if being used. Put two pre-soaked blotting pads in the bottom of the electrode, then the first gel sandwich, with the MEMBRANE SIDE UP. If transferring two gels, add another soaked pad and the second membrane sandwich also membrane side up. Add enough soaked blotting pads to be ~5mm above the top of the electrode. Hold lower electrode above tank, apply upper electrode, squeeze together and push into tank. Seal in place with holder the electrode assembly should be leak proof. Pour in enough buffer to just cover the pads, then fill the outer chamber with distilled water. Put lid on and transfer at 25V for 1-2hrs (current should be ~100mA) Remove membrane, rinse 3x with water (dont skip this step) (Not for fluorescent detection) Soak membrane in Ponceau S solution for ~1 minute Meanwhile, wash the pads, squeeze out and leave to dry (Not for fluorescent detection) Pour Ponceau S back in bottle and wash membrane with water until the water no longer turns pink. If desired, store membrane at 4( in TBS for a few days before probing Soak the gel overnight in Coomassie stain to visualise residual protein use designated staining tray (will be blue!). Next day dispose of stain in the sink and wash gel repeatedly with water to destain. For fluorescent western, be very careful that no trace of Coomassie is transferred to the membrane (use different gloves!) Probing the blot for fluorescent detection Notes: The membrane must not be exposed to Tween until AFTER the blocking step Do not touch the membrane in the vicinity of the bands Use designated plasticware for the antibody incubations Some antibodies, eg from CellSignal have optimised conditions for each antibody follow them, but keep the volumes as in this protocol. Do all tray incubations on a shaker and tube incubations on rollers Two antibodies can be utilised simultaneously with this protocol, provided they are from different species. Use appropriate secondaries to detect in the 700nm (CST 5366) and 800nm (CST5257) regions since the Li-cor doesnt detect the ones normally used for IF. Dont stain the membrane with Ponceau Be careful with Coomassie as the slightest trace on the membrane shows up in the fluorescent detection Block membrane with 5% milk in TBS at room temperature for one hour or overnight at 4( Replace blocking solution with 5% milk in TBST containing primary antibody(s) I normally do this in a 50ml Falcon tube on rollers, in which case 3ml of solution is required. Incubate for 1 hour at room temperature or overnight at 4(. NB: Some antibodies, particularly from NEB, should be used in 5% BSA in TBST. If doing this, wash the membrane 3x 5min with TBST before applying the primary in 3ml in a Falcon tube as above Rinse the membrane with TBST then wash three times with TBST for five minutes. If doing multiple membranes with different antibodies, wash them separately, but they can have the secondary antibody applied together if convenient. Apply the secondary antibody (or antibodies) in TBST with 5% milk for 1hr at room temperature. Use LiCOR secondary antibodies at 1:20,000 (but check!), we keep these antibodies aliquoted at -30, the in use aliquot is at 4deg in the fluorescent antibody box. Cover the plastic box with aluminium foil during this step. Wash membrane three times with TBST for five minutes. Wash once for 5 minutes with TBS (no TWEEN) to remove residual TWEEN. Cover the plastic box with aluminium foil during these washes. Scan the membrane on the new Licor CLX. You can scan it wet (pass the roller over it) or dry (dry is more sensitive)-it has to be fully dried by letting it sit in the dark on 3MM paper at RT for a while or by placing it in a 37( incubator for 20min on 3MM paper covered in foil. Care => this stupid Licor displays 800nm in green and 700nm in red. High sensitivity protocol* As fluorescent protocol, but: Block with 10ml Odyssey Blocking Buffer (Licor) Use primary antibody in 3ml Odyssey Blocking Buffer (Licor) + 0.1% Tween 20 Use PBS-T for washes (PBS+0.1% Tween-20) Use secondary antibody 1:15,000 in 10ml Odyssey Blocking Buffer (Licor) + 0.1% Tween 20 *use High sensitivity protocol when working with low cell number. Probing the blot for chemiluminescent detection Notes Some antibodies, eg from CellSignal have optimised conditions for each antibody follow them, but keep the volumes as in this protocol. Do all tray incubations on a shaker and tube incubations on rollers Block membrane with 5% milk in TBST at room temperature for one hour or overnight at 4( Replace blocking solution with more 5% milk in TBST containing primary antibody. I normally do this in a 50ml Falcon tube on rollers, in which case 3ml of solution is required. Incubate for 1 hour at room temperature. Rinse the membrane with TBST + 5% milk then wash four times with TBST + 5% milk for five minutes. If doing multiple membranes with different antibodies, wash them separately, but they can have the secondary antibody applied together if convenient. Apply the secondary antibody in TBST with 5% milk for 1hr at room temperature Wash membrane four times with TBST for five minutes. Wash once for 5 minutes with TBS (no TWEEN) to remove residual TWEEN. Pat the membrane dry, and apply 3ml SuperSignal West Pico to the membrane Leave for 5 min then dry on tissue paper and wrap in Saran Image using a gel doc system or film Stripping the membrane 450(l - Me 5ml SDS (20%) 3.125ml 1M Tris (pH 6.8) 41.425 ml H20 50ml falcon tube Soak the membrane at 50oC for 30 mins, then wash 3 x in 1x TBST. Block the membrane as usual. Staining the membrane Membranes can be stained after blotting, but can no longer be used for fluorescent detection: Amido black: Stain 15-30min with amido black solution, destain 5-10 min repeatedly with amido black destain Ponceau: Stain for a few minutes with Ponceau solution then rinse repeatedly with water. Solutions 2x Protein loading dye: 100mM Tris-Cl pH6.8 4% SDS 0.2% Bromophenol Blue 20% glycerol 200mM DTT (add fresh before use) Urea Sample buffer: 10ml: 70mM Tris pH6.8 700l of 1M 8M urea 4.8g 5% SDS 2.5ml 20% (Biorad stuff) 1mM EDTA 20l of 0.5M water to 9ml, warm gently to dissolve 100mM DTT Add from 1M just before use 0.01% Orange G 10x Tris-glycine running buffer: 30.2g Tris base 188g glycine 10g SDS dH2O to 1L 10x Transfer buffer: 7.28g Tris base 36g glycine Water to 500ml Dilute to 1x in 20% methanol final just before use NB: Currently 10x TBS is made by media service, so just dilute and add TWEEN. 10x TBS: For 500ml: 15g Tris HCl 40g NaCl 1g KCl Add dH2O to 400ml Add 10ml conc. HCl Check pH, adjust if needed to pH7.4 Add dH2O to 500ml TBST: 1x TBS 0.1% Tween-20 Coomassie stain: Mix 200ml ethanol with ~700ml water in 1L bottle Add 18.8ml phosphoric acid (85%) Add 80g ammonium sulphate Let this dissolve with occasional mixing (takes a few minutes) Meanwhile, dissolve 0.8g coomassie Brilliant Blue G250 in 50ml water in a 50ml Falcon tube. Ensure this is fully dissolved (shake well can you see residual powder when you invert the tube?) Add the coomassie solution to the bottle and add water to 1L Ponceau S: 0.1% Ponceau S 5% acetic acid Amido black: 0.1% napthol blue-black 10% methanol 2% acetic acid Amido black destain: 50% methanol 7% acetic acid  Amounts of APS and TEMED vary with gel percentage, see Sambrook and Russell Appendix 8  Samples with high concentrations of urea should not be chilled on ice as they tend to freeze.  We use Millipore PVDF membrane certified for fluorescence for fluorescent westerns     0Ulmn     ` a c p q  6 = > ? M a c g v ùõìñױר㱨߱h# jmhA-hA- hgMH*hmn    > ? gd;$a$gd&@{ % ) * + 4 5 E F G f g JKLm89:BCDELzɿɶhS"`hS"`h&@{5 hl#m5 h{5h#jh!,i0JUh&@{hh-h!,iOJQJh!,i jhlhA-h#-h;h;hlhh> hGhGh"hQ jh. jhGh.h}9hcG hG h{hG hG5D,;{|vw !!^gdQ^gd.gdG x^gdQ x`gdQ !!>!?!`!!!!!!!!!"""R"S"c""""""##Y#Z######7$l$q$$$$$$$$$$$$$$$$$]%f%%haohrkhhA- jh;h;hS"`h8h_'hGh{ h{h{ hG5 h{5hEIhl#mhhBih]h]hEI5 hl#m5h]h]5;!>!?!!!!!""R"S""""##Y#Z###$$$%%gd`gdG^gdGgdl#m^gdBi`gdBi%%%%%% &D&P&Q&R&V&s&v&&&&&&&&&&&&&&''''(,(.(R(^(t(v(z(((((\)^)))*$*%*********ƻݶݱݭhwhw5hw h. h. h++j h. 5h. h {H* h {H*hgh!,imH sH hgh {mH sH  jmh { h {5h { hEIhEIhaohhA-hrkh;hS"`9%%%Q&R&&&&&&&''(,(^(z(((\)^)))$*%***** `^``gd. ***+%+&+/+1+K+\++++++++++ ,6,a,t,u,v,z,,,,,,,,,,,,,,,,,,,,,,,,-- ---- -!-$-1-3-A-G-V-W-]-^----󵵱hfh#-h2h-h8h hehe heH*hehaoh1 hVEh1 hgh1 huquhwhwh5D**&+1+K+\+++++++ ,6,a,t,u,v,,,,,,, - -W- p^p`gd1 gd1 gdwW-^-------..A.X.Y.g.w.x....X?X@XAXBXCXDX׸ huquh-h>0JmHnHu h0Jjh0JUhH h"hhmHnHuhUjhUjhmUhmh@@-jh@@-0JU% FILENAME Western blotting v1 7 v2.2 Houseley lab  PAGE 4 ,1h. A!"#$% x2 0@P`p2( 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p8XV~ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@66666_HmH nH sH tH @`@ NormalCJ_HaJmH sH tH DA`D Default Paragraph FontRiR  Table Normal4 l4a (k (No List 4@4 ;Header  9r 4 @4 ;Footer  9r .)@. ; Page Number<+"< * A> @R>  (BO)MBT.$@0H!A>풠Uc-zD[&!rX=}zC0` ި%.]Ssd--7 +fOZեrŵVœ\lji2ZGwm-3˵j7\ Uk5FҨ-:xRkcr3Ϣ+9kji9OP Et-j|#p;E=Ɖ5Z2sgF=8 K}*7c<`*HJTcB<{Jc]\ Ҡk=ti"MGfIw&9ql> $>HmPd{(6%z:"'/f7w0qBcF6f Iöi1(\}B5ҹ~Bcr6I;}mY/lIz1!) ac 1fm ƪN^I77yrJ'd$s<{uC>== Ƌ(uX=WA NC2>GK<(C,ݖm: &-8j^N܀ݑ$4:/x vTu>*ٞn{M.Ǿ0v4<1>&ⶏVn.B>1CḑOk!#;Ҍ}$pQ˙y')fY?u \$/1d8*ZI$G#d\,{uk<$:lWV j^ZơSc*+ESa1똀 k3Ģxzjv3,jZU3@jWu;z \v5i?{8&==ϘNX1?  O4׹ӧCvHa01 %xz24ĥ=m X\(7Xjg !Ӆqd? cG7.`~w*?, 2 nN*"Fz_&n &\ F:l[+%f `J^(Z^(IZ BBOOOR G %*-1DX!#%(* !%*W-f0DX "$&') -CJLR!8@0(  B S  ? u{v|EGMWv#19D!"$$p%y%&(&&&& (((((((((\(_(hl7=|47X[ / 2 (3$+CIQX#01I!!x"{"""##n#q#$$0$3$m$r$ %%Q&S&&( ( (((((((((\(_(333333333333333333333333333333333333333333333!$8$&&& (((((((((\(_(x|& ((;(?(@(P(Z(_(-wem]E N $ KcG H b%_'@ +y,#-@@-2Mc2wu5xx>ϰZ=ɱU:;"YYO~R YGQ ^#!_W[A-1 |}9#9g0gM"`r -{_S-)-Uutp,P;&&@4 (^(@(T@@UnknownG.[x Times New Roman5Symbol3. .[x Arial7..{$ CalibriC. Aptos Display3. AptosA$BCambria Math"qh|G ,!F,!F!20&& 3Q @P ?l2!xxb+ Western blottingJonathan HouseleyBaptiste Piguet Oh+'0  < H T `lt|Western blottingJonathan HouseleyNormalBaptiste Piguet26Microsoft Office Word@9[@\`@ޣ_@|c!y,! ՜.+,0  hp  Edinburgh UniversityF& Western blotting Title  !"#$%&'()*+,-/0123456789:;<=>?@BCDEFGHJKLMNOPSTWRoot Entry Fp\&yV@1Table.%WordDocument .ZSummaryInformation(ADocumentSummaryInformation8IMsoDataStorepZ&ypZ&yW02ZYUKGUHZUN==2pZ&ypZ&yItem PropertiesUCompObj r   F Microsoft Word 97-2003 Document MSWordDocWord.Document.89q