פרוטוקול מושלם לביצוע ווסטרן בלוט כולל את כל השלבים.
מומלץ להוריד ולשמור במחשב בתור קובץ כדי לראות בצורה מיטבית את המלל ואת העימוד מכיוון שהאתר לעיתים לא מציג אותם טוב
פרוטוקול מושלם לביצוע ווסטרן בלוט כולל את כל השלבים.
מומלץ להוריד ולשמור במחשב בתור קובץ כדי לראות בצורה מיטבית את המלל ואת העימוד מכיוון שהאתר לעיתים לא מציג אותם טוב
פרוטוקול מושלם לביצוע ווסטרן בלוט כולל את כל השלבים.
מומלץ להוריד ולשמור במחשב בתור קובץ כדי לראות בצורה מיטבית את המלל ואת העימוד מכיוון שהאתר לעיתים לא מציג אותם טוב
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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 30amroran 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
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TAA TO NCAP? pr KMD FyONT TAPIA a’ 2S} OM anna pin AoA Mk PSA? 12 *
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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
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KCI-2
‘Na:HPOs (anhydrous) - 14.4
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pH7.499 a> -
DDW oy 1000ml 9 awa? -
(qm AYN WIA» M2) APINB TBSx10 M237
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NaCl - 87g
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pH7.6 59 ay" -
DDW ay 1000ml > pwn? -
ami mow TBSx1 non
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0.45ml — 85% Phosphoric acid
0.8ml — Coomassie stock solution
40ml—DDW
10m! - Ethanol
mp7 10 waa an?
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Ser Brilliant blue (FlukaG250 #27815) + 100ml DDW
AC atv?
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SOUR PN -
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APSA WN? NIM NVI? APIA
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MINNA NX NWAY ANY YP PAPW MTD NPN Sw MW NID PyOAD prove Ya NWI 993 -APOT
wav? nae
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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 wateramagn yom
M2217 99 vortex + spin down ys3> (1
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abapn ‘s> manana vom oy bw BSA 7 ment nven 3
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(801 UPW
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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
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(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 wellssom 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) S979repwranw 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
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