24小时热门版块排行榜

>论坛更新日志 (3914)
>虫友互识 (1226)
>文献求助 (321)
>导师招生 (209)
>休闲灌水 (191)
>考博 (164)
>教师之家 (103)
>硕博家园 (95)
>博后之家 (89)
>论文投稿 (87)
>找工作 (78)
>基金申请 (74)
>论文道贺祈福 (68)
>考研 (65)
>公派出国 (56)
>绿色求助(高悬赏) (46)

返回列表

当前主题已经存档。

当前只显示满足指定条件的回帖，点击这里查看本话题的所有回帖

zhsteel

金虫 (小有名气)

应助: 1 (幼儿园)
金币: 1171.2
红花: 2
帖子: 216
在线: 42.3小时
虫号: 223866
注册: 2006-03-22
专业: 生物化学

[交流] SDS-PAGE

SDS-PAGE
Materials:
• 6X Sample Buffer (store aliquots at -20 deg C):
6X Composition (for 10 mls)
60% Glycerol 6.0 ml 100% Glycerol
300 mM Tris (pH 6.8) 3.0 ml 1 M Tris 6.8
12 mM EDTA 240 ul 0.5 M EDTA
12% SDS 1.2 g SDS
864 mM 2-mercaptoethanol 600 ul 2-mercaptoethanol
0.05% bromophenol blue "pinch" bromophenol blue
• for polyacrylamide gel:
1 M Tris-Cl (pH 8.8) 10X Electrophoresis Buffer
1 M Tris-Cl (pH 6.8) 30 g Tris
20% SDS 145 g Glycine
40% Acrylamide (37.5:1 acrylamide:bis-acrylamide) 10 g SDS
10% Ammonium persulfate (store aliquots @ -20) bring to 1 L with H2O
TEMED
2% Agarose
0.01% SDS
• for staining/destaining gel:
Coomassie Staining Solution Destaining Solution
0.1% Coomassie brilliant blue R-250 50% Methanol
50% Methanol 10% Acetic Acid
10% Acetic Acid
Gel Equilibration Buffer
(dissolve Coomassie in Methanol before adding water and acetic acid - will require stirring 1-2 hours) 20% Methanol
10% Glycerol

Procedure:
1. Pour Polyacrylamide Gel
o assemble gel sandwich
 wash well and rinse (final w/ 95% EtOH) plates, spacers, and combs
 lay back plate (cut out plate) on flat surface
 place spacers along bottom and sides of back plate
 place top plate on top of spacers offset from bottom edge by 1-2 mm
 clamp together with 1" binder clips
 seal sandwich along bottom and sides with molten 2% agarose
o pour separating gel (5-15%)
 determine percent gel needed and combine components in 15 ml conical tube
Separating Gel Percentage (vol in mls) final
stock solution 5% 6% 7% 8% 9% 10% 11% 12% 13% 14% 15% conc.
1 M Tris 8.8 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 375 mM
20% SDS 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.1 %
40% Acrylamide 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 5-15 %
H2O 4.95 4.70 4.45 4.20 3.95 3.70 3.45 3.20 2.95 2.70 2.45
o add 100 ul 10% APS and 6 ul TEMED (increase to 10ul TEMED if gel percent is less than 8%)
o mix well by inverting tube
o pour solution into gel sandwich using a Pasteur pippette (run it along a side spacer) until desired level
o leave enough room for a stacking gel of 1/2 well depth below the level where the comb teeth reach
o gently overlay with 0.01% SDS to create a barrier between the gel and the air
o allow gel to polymerize 30-60 minutes at room temp or until interface appears
o pour stacking gel (3.75%)
 pour off aqueous layer from separating gel and rinse with ddH2O
 combine components for stacking gel:
 2.329 mls H2O
 0.375 mls Tris 6.8
 281 ul 40% Acrylamide mix
 15 ul 20% SDS
 30 ul 10% APS
 5 ul TEMED
 pour stacking solution on top of separating gel
 insert comb into stacking gel taking care to avoid forming bubbles on the ends of the teeth
 allow gel to polymerize 30-60 minutes
o clamp gel onto electrophoresis tank
 carefully remove binding clips and the comb from gel
 remove bottom spacer (leave the side spacers)
 place gel against electrophoresis tank with the cut out plate facing the tank and clamp into place with large binding clips
 fill upper and lower reservoirs with 1X Electrophoresis buffer
 straighten wills out with tip of Hamilton syringe
 rinse out wells and bottom of gel with buffer using syringe with bent needle for bottom edge
2. Prepare samples
o thaw protein samples rapidly in room temp water bath
o add 1/5 vol 6X Sample Buffer to protein samples
o boil samples for 5-10 minutes
o spin down samples in microfuge for 1 minutes
3. Separate protein samples by PAGE
o load samples into wells using Hamilton syringe
o fill empty wells with protein extract buffer + Sample dye
o attach electrodes so that proteins will move towards the anode (+ or red lead)
o run gel at 100-200 V until dye front reaches the bottom of the gel
4. Stain gel to visualize protein bands
o disconnect electrodes, dump out electrophoresis buffer, and remove gel sandwich from tank
o remove side spacers and carefully pry apart the plates so that the gel remains on one plate
o pour staining solution into pyrex dish
o invert plate with gel into the staining solution and gently allow gel to "float" off of plate into solution
o cover with plastic wrap and gently agitate gel for 60-120 minutes (longer will increase sensitivity but requires more destaining)
o remove staining solution (can be reused many times) and rinse gel with ddH2O to remove excess stain
o add destaining solution and agitate until satisfied
o change destaining solution and agitate until proper level of destaining is achieved (can destain overnight if needed)
5. Preserve gel
o remove destaining solution
o agitate gel in gel equilibration buffer 15-30 minutes
o soak two sheets of cellophane in ddH2O
o place one sheet of cellophane on gel drying rack
o place gel on top of cellophane, taking care to avoid trapping air bubbles
o place second sheet of cellophane on top of gel and remove any trapped air bubbles
o place upper rack on top of cellophane-gel sandwich and clamp together
o allow to air dry overnight

Notes & Miscellaneous
• Coomassie staining sensitivity is approximately 100 ng protein
• unpolymerized acrylamide is a neurotoxin - wear gloves when handling
o once polymerized, gels can be disposed off in the regular trash
• keep excess solution to monitor polymerization process
• to facillitate visualization of wells in the stacking gel, add bromophenol blue to stacking solution
• add KimWipes to destaining solution to soak up dye as it diffuses out of gel (if left in too long, i.e. overnight, can also cause destaining of proteins themselves)
• glycerol in equilibration buffer helps prevent cracking during drying of high percentage gels
• MW determination:
o Rf value = [distance of protein migration] / [distance of tracking dye migration]
o plot log(MW) vs Rf for size standards and interpolate unknown protein from its Rf value
• offsetting top plate of sandwich allows easy rinsing of gel using a bent 20G needle
• insert comb into stacking gel at an angle to help avoid trapping air bubbles at end of comb teeth
• "smiling" and "frowning" of gels largely due to unequal heat distribution / salt concentration across gel
o run gels slower or with cooling in the cold room
o fill empty wells with extract buffer

回复此楼