You are on page 1of 13
ViTs wybs peo — ype Bio Rad mini protean v Duupa Dan by ypns> ona by 2217 (DDW) ovnys orp om AWW 70% NN OYIM NDT NK MPI? * weary mani wx Kimwipes am napen (8 qrowwa oy ow maswa oy NW Taw ONTND FUvVAA Fw PINT TNs AYIA NR OW (1 omnon “ay AK NOT spor mua? *1> op pA Iw NVI! PI WN Mn wT Va OPSLAND DW? WI - ers WY TAY DONA ON POT ABD AN DTD AVDA ny ow oso — AT APTA (2 Apna Ya 73 37a nt ON ABP FT wr ON 95°C 2 ouenn nove/oaNT ne pT TINA TweDA? ona ATA MeANT NN NNT (3 mia nga? Tes ——_— (1 npoa) pnnna 9x71 nx ran (4 Tm > — (ravana “nna pha) pra waon bw Wyn pone pon’ ww nana Ya ne oye (5 ‘yom yrs moran 77D) Y's NN Tw) MTD ANA TWDrs DY OX? DDW 400p1 > Derr (6 (ora yen ainnnna ,naveyn anger) Pera os wanway Wa or (om ena yaw *Y28 WwpNA YA dy APY na 959 NYT? ya) AWPNA S'sAw MN? (7 Janna ws a napw Pen mann Y9.na5w Yap) uamram 1x ,D°aT Nk BrDETN ypNan Ne ITT (8 3ml >= rr>t3rn bw Tym 1p dyn wD oT doa AN OSU (9 Ara negra pray "7D ,2°e Oy 2nan w ava ponA ne PIO Wea NW APIA NN NW? - SMR 2D? TN DBD AEN NW TD TWP TET OX? PIODT NX OTT? (10 eprom 72a dyn Yan bw VAN ne a (11 -VPATON UR? prONT NK ONoETD ,AWyANA d'sAw MK (12 NYTWIT NX Mp? "7D TVD MyTN2 Runnig Buffer oy Y's MN wae (13 DTD DY YMIN WY YD FANT NW NW IE? NP MINIT ON TT sD rip apMy OIA IAN THRW 7D AN aN MPva ANY! ADD TP MAwATY awn * spina menart musa (2 (6 np0a) THETIA NPrTAA PIMA ND MNsIN sy MANTA DN PAY (1 .S.Bx1 By 18 ,7512 ON MINDIITA BY Sample buffer (S.B) 7.8 773 * 95°C mp7 - Sample buffer + paynm nx mand (2 Sn Merny) APM MxwaTTTW TW MPT 10 -2 Mon? (3 (fasona prone amy hoa mNeT TH MAP MRITTA WD *) <3 npora Winns ASNT WpNa MS ows Yan OY NVSTT ne OEM (4 mayonm 70 nx yy ow? - awn (5 18a ypMan nN waOIIA Naw NMP "1D “VOD AEA ypnaA yw AODNAW AKOTA 36 PrTa» (6 SINT Typ OY DINK DY MysOND pyUA? .wpraan padnA MA sb WPA NAD (7 enepn MYAWAA NNN WPA Syl nwa 10 9y propa) neawa oa 45pl ena? Ty — MNMATTA NN ANAT OR? PUT (8 1.5mm >y 3193 Fysyn Mp}? Onan TWwRD AVA yA ne TW! KAA nnn? eA ne ODA * sip PRAT DMNA TAPAT NN OR? TINY “PUT Aaa a IM OMA? NEDA TMT Je waa AMMA PIAA AW XY MITT Siar TOD MNDATTA DN PRAT TN WY ON MP NINA WP NID — ND AP VT AT? * Bx LW .Mpov MNT OTA MPA NV NAA PCAN of poo yw) nvopA bw owas DvakIn TODA yy OraDA IRD - NM? (9 (Miliamper) syap Ont 38 ,(Volt) srap nim bw awsa mon po nN MyEA? 12 (10 <«T1OV-39 “van? panna 939 mrss maaart7 Wwe 9OV a PaNAD (11 Ayn 999 9o"0a dn Mennng nya dw oy nw" APN AEA nywanD DR (12 Fann x? — (4 noo) oonaona 999 Nx nyIEW *ONDIP NAS ASTAA POD HEI? 1 (13 4°C 2 ow (T.B) Transfer buffer msraa Bro ivy? PaA? (14 prpon PR yom oma APIA by Foy AAW DoAN Y'sn TRY abn dw TAD TIN PR ON * JTW BK FONT HEPA MDT Mano Ip? dye KuD PAA RM < APIA PAIN mann) m7 (3 4°C 2 T.B 2 qoona nen ongon AMET NN OW? - ETNA OVO 739 MPT 10-9 (1 sua, aT NR Py aw? NP MIA ATIANA NE Pavey * T.B oy mypa ow’ 9a nx nro wenn (2 hones wan on NNR — T.B nomena omy ne ove Te DAT Iw AR TT wP fore Upper bw pona nx prot jinn (3 ango 227A x T.B Non (10 (ori? ON) DMB OANA *BP AODIT AK MD? (11 .200mA-23 MIPT BWI AVY wea 37KD ora Nn yA? (12 nay) yor any np? 91a APT bpwa ya NIT * man ypne | TRS Tom ne JANA Posy an Yan 99 IPI kw? OX TANT ores * cponabnin Yaa) mrpvapo NY mina — wren mraz (7 nonin maaan nx ex (1 1273 (Ponceau) 1°ond NOwN 7209 (2 aranNA yy MTVU Tow? No wND DB *799 mITNNA NN MV? (3 syeonen nx pon 3b anna Tw? Wy OvIRD ONS OMNIA ME ONT WH (4 ain vista? 0 DDW 3 7ee9 (5 (E277 8) IpIIT bw OY oTaT nN MINTS BVA AIINAA by OWT (6 JFoinn 1" mpwa neva ANwNT Ne ww (7 -mxsnna dya7y Ts? AIDA NN PAD? (8 amranen ve pens Drpon Tn (9 aowwan przona ya gy mT ANNA NN Tin? wry AND owaNTD OX (10 span) "9D MME PSD OAD NA PO By TINNY ,rapHa MUAIwD m2 By OW BR (LL maraaan p23 28 PR TW DUP DSU 29a PBS-T oy *239 nEWNT Nx TINA? (12 nyt) mm yma Xd) by? vaEA NINN ADIVNT NN CoM "2B PBS-T a pow? No — fs STN 7K serin TBS-T 3 o3 wana? ya PBS-T 3 wis wr raw Dye 922 MwA amon (7 BSA i8 skim milk 1%-5% 2"73 meh 9M3 BY 32 MPT 60 OW - ana "p02 drew (skim milk 2.5 gram in 50 ml PBS-T = 5%) JI IEW TBD TMV 9M3 IMM 25% 2 pI? - Jo prRmay maw AVON IM NVA? VD I 9D? - 4°C 3 over night mon v2 03 13 - Ay ( OR (2 (pon ww ,4°C 3 (17Hr) over night ,yo33 po7za voy’ [pra oy yaa AANA nN ow (3 Syyeooa (yaa Aon) IMA rTM Su Sml > 10% Sodium Azide 5 SOul nsoiaa “win wary? pan nN ew ya ODA (4 APC 3 pon 73 (avon 9298 PBS-T oy 1772) IL ya (t ycyua PBS-T 3 SMin mowy 5 (1 -RT-2 70702 LHe wea I 3 (2 dyv>va PBS-T 3 SMin mavww 5 (3 SMR NH wD THe? JoUT AND IW PI WIINa ANIA NN Ovo Wav? 13 — MAT (5 n503 789) stripping ame ( D7 pros Wann 1k ,AIMD PaTDBA IR TIP ODD MMA wanwT? WOR - nen ‘poprna 738 Enhenced Chemiluminescense (ECL) :-nxn naan noap poan = 3 asin Doap rama wPOPND why wa TN? TarsaW HRP OKT 721 (202) ovgo gin na (#7 oro) AATaAN WINNT Na 932 PN we ATA tT APAT AMR WR DW FTOMD NIT PNT WIND TY TDN AUK NII TAY VND PIRI NWT I Dip "2a OY DNDN OTD UN SPT Mn AP we DA TN DWNT yor way IMs oN 2 ows TI - mnona mms (In comer penn nx yu’ nora ns 92 (1 ufpynp a 312 — nmonn nx Svar (2 SDITN TS PT TRWT? TNT AN MII? (3. wpa orpiapan vw ow 1:1 by ona ECL par? (4 stam 729 ¥82 WI — ECL novan Sml es nnosa naan? vow (5 saanaee + ECL "pt 3 9393 (6 NOX mm nosypIy ADIN py pW Ta ANAT NK Ow? (7 Senna zmsrwn mmo noi?) ETA AS wha yor? MDIVA NR aon AMIVAA dy or. Ow (8 (377. DX PIT? WK PIN pr|ON PaO PR ON AEN yor MayAIN APT by Myp Aawh oy 8 Ns NX Mat ow pyr ne Nyy ree Iw eo OA Dw APE nx Yep> lawn (7 DUAN API Pw WO WA AY WRI NI? pmonn dw den Twa ABWhA MN? oxen nN peUA? (9 38 DRT NR pr aTY ya pop THN pI naaN ANE X a1 Sy ANTI AMA Ie Mand (10 DPD Ty py? Javan Drea eww Ty MPT 3 2 pnaT (10 “_RBNOM Por AK abn BW nN Be oy BWI? (1 orm mime (Qn -DYTN TS PT PRWA? WRT AR MI (1 spa pp iapan aw oe 1:1 9 oa ECL pon? (2 stage 729 9329 WI — ECL novan Sml mes nnosa nanny own 3 sarge + ECL "p73 vv (4 JRTON MM NosrpN AINA pyea Mp! TNA AIAN ne Ow» (5 Mptwn map prow) ETA MD WhA jot noDMT AK Tuo AMaBAT by OND ow» (6 pm pron 2a70 PX Ox TBTwHA Tor Mean AVI Wyd Iw ASP Aw OF 3x MwA NN MARA Tey pron NX neTy A IW oe ODA dw APR nN y9p> lawn (7 DVM API Pw PO WA ADD WRI NI? -Developer novana nv3y 7733 (8 cpmea naw ma (9 ‘enna ore ovvs) orsnw ww Fixer novan (10 Roun yor en padHA BY Ne avr|n dy OWT? (11 .BY' pea NPM BT oman pws ( bum om prod ,ommie3 oremnim Bx (1 imaged nisin mywnxa main m3 (18 sto? Thunpag muDIA ne arna Pena TA? Wy http://rsbweb.nih.gov/ij/ prion oan yw mavans pap nk mns> File>Open(1 Process>Subtract Background (2 Edit>Invert (3 cnyn mya 29 PAs BN Image> Adjust>Brightnes/Background (4 proinn os oan WWW Yow prTab ana drwan TAMA NN Pao Ma BrPaA BIA TWN? (5 sama pn? mara Analyze>Measure (6 Results>Set measurement>Mean Grey Area nx 720? Results 1n3 (7 ow now Tan Mew nN aw OTD STA ST nN maw a AN way NIA AS OM (S -6 7903 W22 Measure 9y ABN?" NIT File>Sellect All mxsin7 973 (9 edit>Copy (10 -MNSINA DN 7OPND PITA? (11 Sonar an mean oy ae vA pram pan dw nwa oy pywA mewn OY Tw oy (12 mood Joo NMA wav My 21 MWA Ms1aPA Pa ANA XW TA - house keeping gene ) .Qonen nsvagn as 7232 03 wonwa> WeR .praw ,PUPX :ANT? AIvOTTY NA spans Yw mayen new] nyo Mp? v7 SaTwaA pA PIAA TT NN orp (13 Doyenyn DAA’ DIT AN PITTA DOD < EPW-MNSY aA Sy AAMANA NN pene 13 (14 Ju7np-TNNDI Past 3"nN1 Copy To System Jel Pro main myzmea (20 Son nx opm — Lanes m3? DEW DANA NS Ov ~Add lanes -TN7P -Mass lod por Twp -lod Sopa mnesinn nx 8s" "79 area YopxA Pw AavEAA yaIP MX “ANID options< File - Sopx? niavan mun nx “Pay — DD to Excel pray pan amg yaa meee ew paAW Pre) MO WNT BP Iw ANTONY AAT MAT OVW - prog rw 93959 ory Oy pra Ta OPIW oy Brow AAAI MN TANT AN Ie (iu Yon B ATID NIA DOT kW yw MMV? OXNAD MIMW WI OB OMA woNs? DAD WH - cavum pao msn mown nee" SONIA BAIN WONT WPI PBA MP Ps om ow = on now m7 msI = aM ness 729 TDI — yPT-T MA WI - yOpNa.UTm sheet MNS? w ,o-wW “IS? NOW [ean by MENINT NX ABW Tan DIN - -20C a 7unw’ rN? war — TEMED a nwnp? vWor'v07 — APS - Ammonium persulfate an nwnp’ orn - TEMED Jha wry "1p “rw wna oBIa%nA NX gow — SDS pH nm yy aniw ~ TRIS ap" niga — Ine 7 pH 1 no" yar nivtaay Lower (resolving) 1 Upper (Stacking) maaw ‘nwa *u2 937 pine meniahn? DY Aa (pH 6.8) psa dy AMwpPeIPR MPI? oXMD esa Ya yy pH ‘open 79 by men vyann mnnnna naswa zmaswA pa NAA npr ra wai TNR apa spabnn prva? oyna pabnn na 93 arma panna 937 No ne - any asp pina ww Bor sro Pan Ay PTW 490 - MD mavpa oNaAT— 9797 95." ON - oypm pyran ornaon — yey 93.0" OX - emabnn rma 3b pone pinnn a noe may vase 399 - Soames | payna ym. mind WW 7.5% | _76K-225K 10% | 31K-76K 12.5% | 12K-31K. ros ornona (era 3) mms) NANT Pre % 7.5% 10% 12.5% 15% 40% 3 3.75 5 6.25 15 Acrylamin(ml) Tris 1.5M pH 3 3 5 3 3 8.8 (ml) 20% SDS (ull) 100 100 100 100 100 DDW (ml) 118 11.25 9.8 85 15 10% APS (ul) 120 120 120 120 120 TEMED (ut!) 20 20 20 20 20 5% Wy Ds pase] ppm: | sew | oan 40% 0.625 1.25 1.875 Acrylamin(ml) Tris 0.5M pH 1.25 25 3.75 6.8 (ml) 20% SDS (ul) 25 50 75 DDW (ml) 3 6 9 10% APS (jt!) 30 60 90 TEMED (ul) 10 20 30 amroran navn — 2 np (qa3v9 ao WNIT I) WONT 0.1% in 5% Acetic acid Ponceau s — Amresco ~ 0860 0.05gr in 50ml 5% Acetic acid 5% Acetic acid = 2.5ml Acetic acid Glacial + 47.5DDw Running Buffer x10 Tris - 30gr Glycine - 144gr DDW - 1L> o4wa? AC a aw Running Buffer x1 wrewn 352 PT? Running buffer x10 — 100m! SDS10% - 10m! DDW- IL> own? -Running Buffer prav2r weew y2v2 Tris\glycine\SDS X10 2) wr mTayEa cw) 1 bw nbs DDW by 10% orm Transfer Buffer JANTAN BVO ANP? OMB ya 7 PIN? * TAA TO NCAP? pr KMD FyONT TAPIA a’ 2S} OM anna pin AoA Mk PSA? 12 * yo (@y2a wNY SDS v7 Running buffer x10 3) 1392 Tris\glycine X10- 100ml Methanol ~ 150m! DDW - 1L> o4wn? Transfer Buffer o> 2 pom? pom ayy mu mW TD Sample Buffer X3 1.5M Tris-HCI ~ pH 6.8 ~ 3.75ml SDS — 0.9¢r (sodium dodcyl sulfate) Glycerol - 3ml BMercaptoEthanol ~ 1.5m 0.5M EDTA - 20p1 Bromophenol Blue - Few crystals DDW — 10ml > nu? ome p’svim Ws TMwL-AT Mav EraMA Nx om - reducing agents - BMer, DTT inn 'spua rs mnp DTT ow x? DTT ay > TAD nas? Ts PMer oy - <(mapy DAN WT? 19) AIK PBSx10 ra37 NaCl— 80 KCI-2 ‘Na:HPOs (anhydrous) - 14.4 KH2PO, — 2.4 Jou Dy Pre MIVA WPI Ne ova DDW 800m! FONT - pH7.499 a> - DDW oy 1000ml 9 awa? - (qm AYN WIA» M2) APINB TBSx10 M237 Tris base — 24g NaCl - 87g (bmp DY TED ATV OPI aK oA DDW 800ml porn? - pH7.6 59 ay" - DDW ay 1000ml > pwn? - ami mow TBSx1 non Tris HCL 1M pH 7.4—$Oml ‘NaCl 5M -33ml DDW ay 1000ml > awa? SNOOP WI TBS-T 1x) PBS-T 0.05% 10% Tween in PBS - Stock solution na37 (1 tween 7 nx "nk PBS 7 nx syor7 DN? .36ml PBS > 4ml tween row? SOml nana. any nie wie? 4°C 3 wrong awd a — Asp mM Ny a mVEDT OV aw -stock solution 7 2.5ml tween D°5°079 500 ml PBS -9 133 "HN? (2 -PBS 2 p23 TBS 3 03 wanvay qWweX sya * APS zultra-pure water (UPW) -2 ammonium persulfate np2¥ 10% ann yen naps — 3 ne ww Ann? olay B .9'a7 ay AYIA NK DAPYY DNB! OPN dy ONO IY w> smu? Maman = (E) Buffer Dam preods ov? Ps "Wa Ta WR THN 73 PTW" ON .C 2973? Bro BD ,D Bp ‘899 OV This side towards the gasket 2n2v 17 NN vpn Dw ,A#B) menqa ypnna ova Yan oy ANDINA A OWEN TWwronA HAW ANY (1 brap *999 TAP MDWAA (BoM? Taw yn (C) 9.3) nestor oy AETAA pha ne OIA (2 amb pay NM? oye Tw APIA Pa R.B NID? 3 anypiora naa sn? wea pon R.B x70 (4 -TAVOTA DvaypO NS 7209 71> F pro. Wanwa? Wer NARII MANTA NVA NX VPA? 79 (5 -DwwB pon dyn mx PINAY 1 SoNmIP nyvaz — 4 nor Aamapan 99 eryezey wonse ANW2) Ya OrNadNA ys? MaTEBO NI ANAS SON Soa ne own EAT Iw A (I ivy 1-2- 12.5 Acetic acid + 50% Ethanol 3 7x07" (2 ivy 16-24 -owntp novana ATs 3 (onan yasn nx TA9 *72) MPT 15-20 ws 20% Ethanol oxy — naw (4 ran ama pond aa an 08 49 arn 79 MN MAT? I 732 (5 -zomgh ams prion qa mepu2 mT sn Me BUY (6 DoNDIP nyvas9 TON NII 4gr— Ammonium sulphate 0.45ml — 85% Phosphoric acid 0.8ml — Coomassie stock solution 40ml—DDW 10m! - Ethanol mp7 10 waa an? Coomassie stock solution: Ser Brilliant blue (FlukaG250 #27815) + 100ml DDW AC atv? swamp oy 993 PIA? VIIA NW 9D NK apeyrana) apyree — 5 mp0 Antibody stripping 2 www >"y panma INN were THE? AMaNTA OITNA AR POR? WT buffer sana n>wwye02 P>Ua stripping buffer ov AMaDNA NX MPT 5-15 warMwA - SOUR PN - pny 4 nt toy psa —deze2 mpTS yee DDW aya - (EL ori noon ron win yea) w) ay Aaa wanwAD yore. Savo wr ox apn ECL nowy re oarawen pram ow xm ea avo "OX MPT INR TaN MBO My TA MwA TANT OR TTD cpiabn nnap — 6 neo APSA WN? NIM NVI? APIA 73 32S PSPRM NY DUAN OO BSA DW OW ONIN 72 DVD DIPY OND TD TNs? O.D merp 5 menarra pana a7 ne Map? we 3 AOI MINNA NX NWAY ANY YP PAPW MTD NPN Sw MW NID PyOAD prove Ya NWI 993 -APOT wav? nae BCA Protein assay - Thermo #23227 wp ay yeaa - 7512 O17 Triton) NP-40 9w MINI VBWI We WPA (A-D 9y3n nonpyy parade mA PST? - yao npon? werwy ora OA WS UPW notya panaban meant nx dima? Ws - Pierce #23209 Albumin standard - 2mg/ml 5v mua meaka wanwAd 2 - (roar 290 nN Wire Maven) Dryp TWA OW - capsmiy rao Yea ovoa DIPy PW MATT AON ABN MMS AT Vy WNW WON - vas novsn 200ul + BSA 1x pa9n maT 7w 1Op or 25yl Pw “sro Nos oFVED NINA 723 - WR) saan Ahawn °5 dvan onpy bw BSA a meet nA Final UPW cone Or lysis, buffer Remaining nein! | tube | BSA Qmgiml) | (ml) | Sum~ for use 2000 | A | 86 0 86 93 1500 |__B | 107 36 143; 93 1000 | _C | 93 93 186 700 750| D|30fromB | 50 100 3 300| E/93_ fromc | 93 186 93 250| F (93 fromE | 93 186 15 125[G/93__ fromF | 93 186 3 25|_H[29 fromG | 114 | _143 Blane) 0/110 u4_|_1i4 Sum: 686 286 __ulBSA__2mg/ml__ needed * UPW= Ultra Pure water amagn yom M2217 99 vortex + spin down ys3> (1 sPeaun 2) byon apy bw manasa UPW raven (2 abapn ‘s> manana vom oy bw BSA 7 ment nven 3 + pon 20pl wp ye Wwa) 201 UPW + adn Spl 5° opaW mNaarTA da - aM ND (4 (801 UPW .pvon aon van onpy ny Raa MDATTAD 1 Op - 251 maven (S any 73 mya mv yen 10p maven) anvwan 233 (W.R) vay novan 200p1 neorn (6 eV WH NY TPAD AVANT MR Top TsVT!D Iw (7 AOD 1a TLY|T NN NOI (8 3ToC wWeDPPNA MPT 30 (9 562mm dy bs NA AMR WIM. TUDE MEAP (10 (5°9 povan aon mwas17 Ne wT OX Wot?) va aypy 2 SOPRA PINT NID Bw (1 PX MUDTON NES whan "xls. NI awn? oro3" OWS Jor VOPR PIP Ww * oon ‘pon Noy 2107 52 TwNE ANY wo" A AayUTA) wn wa wenwAD prow mBwT 2a * (npn :(W.R) yagn novwan man - mnpona naa 3.9 + PND 43> 200ul PIB .B vax Sy moe 50° nea? Ts Torna A DUNT NI * 4uil reagent B * number of wells 20041 reagent A * number of wells som mare 27 mp sana Bw man wpa ae Acrylamide (40%) Sigma A7802 ac Tris 1.5M pH 8.8 - lower Bio-Rad 161-0798 RTT (RT=Room temp) Tris 0.5M pH 6.8 - upper Bio-Rad 161-0799 RT-29% SDS 20% (Gan) Amresco 0837 RT-29% Temed Bio-Rad 161-0800 RT-aq7 ‘APS Bio-Rad 161-0700 RT-23979 (Ammonium Persulfate) Sigma A3678 RT-2473 Tris\Glycine\SDS X10 Bio-Rad 161-0732 RT Tris\Glycine X10 Bio-Rad 161-0734 RT-3979 ‘Methanol nna S79 7m 72 WT? Ultra pure water (UPW) | nrxbyamwsn | 01-866-1B RT-2 9% PBS x10 nembyanrwyn | 02-023-5A RT-3 972 TBS x10 Sigma T5912 RT-2.473 (TR) Ponceau s solution Sigma P7170 RT-2479 Skim milk powder Fluka 70166 RT-29% BSA ‘Amresco 0332 eC ECL kit Bio-Rad 170-5061 4C Antibody stripping buffer Geba bio STO10 aC application Nitro cellulose membrane men 10401383 RTT Protein markers Page ruler Plus Fermentas #SMI811 -20°C (rx) Page ruler Plus Fermentas #26616 -20°C Rainbow GE Healthcare RPNSO0E, -20°C (PR) Seablue plus2 invitrogen C5925 4°C (PN) Secondary antibodies HRP-Goat Anti-Mouse Invitrogen 626520 #C (PR) (1:3000) S70 ECL Anti-Rabbit GE Healthcare NA934V 4C (PR) (1:4000) v7 Peroxidase AffiniPure Jackson 705-035-147 aC Donkey Anti-Goat (1:5000) v7" Peroxidase AffiniPure Jackson 115-035-062 eC Goat Anti-Mouse (1:5000) v7 Peroxidase AffiniPure Jackson 111-035-144 aC Goat Anti-Rabbit (1:5000) S979 repwranw una Braw ovwob MATA oUTO - 8 MBO rosa nia D4B8 Don Sw moan 2owa me%Ta sInA? PR https://www.youtube.com/watch?v=b45nSOyPP_4é&index=4&list=PL3264CF81D53 FD4B8 oy sas https://www.youtube.com/watch?v=XUjLO- ck2C8&index=2&list=PL3264CF81D53FD4B8 row) 'PNTION M7 https://www.youtube.com/watch?v=VaXFwfkLdgw spraowa 929 Amat Nw OY PW https://www.youtube.com/playlist?list=PL79DF2FBO8E95D63D neo eT oy To 25.11.14. pan mes meee? raat (9 ne 10 Ey cy} D

You might also like