双向电泳实验流程.docx
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双向电泳实验流程
双向电泳实验流程
●样品制备(Samplepreparation)
●固相预制胶条的水化(IPGstriprehydration)
●第一向等电聚焦(IEF)
●胶条的平衡(IPGstripequilibration)
●第二向SDS-PAGE电泳(SDS-PAGEelectrophresis)
●凝胶的染色及检测(Detection/Staining)
●PDQuest软件分析(Softwareanalysis)
●质谱鉴定(Proteinidentification)
目 录
第一章 实验材料
1.1IPG预制胶条及载体两性电解质
1.2蛋白质定量试剂盒及其试剂
1.3蛋白样品制备试剂盒及其试剂
1.4化学试剂
1.5蛋白质Marker
1.6染色试剂
1.7注意事项
第二章 SDS-PAGE聚丙烯酰胺凝胶电泳
2.1溶液的配制
2.2SDS-PAGE凝胶的配制
2.3操作方法
2.4注意事项
第三章 双向电泳
3.1溶液配制
3.2操作步骤
3.3注意事项
附录1双向电泳完整的操作步骤
附录2 聚丙烯酰胺凝胶电泳凝胶的配置
附录3 细胞样品的一般处理步骤
附录4 组织样品的一般处理步骤
第一章实验材料
1.1IPG预制胶条及载体两性电解质
(一)IPG预制胶条(美国Bio-Rad公司),-20℃冰箱保存,每包12根
项目规格货号原价(元)
IPG预制胶条pH3-10,7cm163-2000799
IPG预制胶条pH3-10,7cm,nonlinear(NL)163-2002799
IPG预制胶条pH4-7,7cm163-2001799
IPG预制胶条pH3-6,7cm163-2003799
IPG预制胶条pH5-8,7cm163-2004799
IPG预制胶条pH7-10,7cm163-2005799
IPG预制胶条pH3.9-5.1,7cm163-2028799
IPG预制胶条pH4.7-5.9,7cm163-2029799
IPG预制胶条pH5.5-6.7,7cm163-2030799
IPG预制胶条pH6.3-8.3,7cm163-2031799
IPG预制胶条pH3-10,11cm163-2014957
IPG预制胶条pH3-10,11cm,nonlinear(NL)163-2016957
IPG预制胶条pH4-7,11cm163-2015957
IPG预制胶条pH3-6,11cm163-2017957
IPG预制胶条pH5-8,11cm163-2018957
IPG预制胶条pH7-10,11cm163-2019957
IPG预制胶条pH3.9-5.1,11cm163-2024957
IPG预制胶条pH4.7-5.9,11cm163-2025957
IPG预制胶条pH5.5-6.7,11cm163-2026957
IPG预制胶条pH6.3-8.3,11cm163-2027957
IPG预制胶条pH3-10,17cm163-20071079
IPG预制胶条pH3-10,17cm,nonlinear(NL)163-20091079
IPG预制胶条pH4-7,17cm163-20081079
IPG预制胶条pH3-6,17cm163-20101079
IPG预制胶条pH5-8,17cm163-20111079
IPG预制胶条pH7-10,17cm163-20121079
IPG预制胶条pH3.9-5.1,17cm163-20201079
IPG预制胶条pH4.7-5.9,17cm163-20211079
IPG预制胶条pH5.5-6.7,17cm163-20221079
IPG预制胶条pH6.3-8.3,17cm163-20231079
IPG预制胶条pH3-10,18cm163-20321079
IPG预制胶条pH3-10,18cm,nonlinear(NL)163-20331079
IPG预制胶条pH4-7,18cm163-20341079
IPG预制胶条pH3-6,18cm163-20351079
IPG预制胶条pH5-8,18cm163-20361079
IPG预制胶条pH7-10,18cm163-20371079
IPG预制胶条pH3.9-5.1,18cm163-20381079
IPG预制胶条pH4.7-5.9,18cm163-20391079
IPG预制胶条pH5.5-6.7,18cm163-20401079
IPG预制胶条pH6.3-8.3,18cm163-20411079
IPG预制胶条pH3-10,24cm163-20421292
IPG预制胶条pH3-10,24cm,nonlinear(NL)163-20431292
IPG预制胶条pH4-7,24cm163-20441292
IPG预制胶条pH3-6,24cm163-20451292
IPG预制胶条pH5-8,24cm163-20461292
IPG预制胶条pH7-10,24cm163-20471292
IPG预制胶条pH3.9-5.1,24cm163-20481292
IPG预制胶条pH4.7-5.9,24cm163-20491292
IPG预制胶条pH5.5-6.7,24cm163-20501292
IPG预制胶条pH6.3-8.3,24cm163-20511292
(二)载体两性电解质(美国Bio-Rad公司),4℃冰箱保存
项目货号原价(元)
Bio-Lyte3/10Ampholyte,40%,10ml163-11121704
Bio-Lyte3/10Ampholyte,40%,25ml163-11132463
Bio-Lyte3/5Ampholyte,20%,10ml163-11323275
Bio-Lyte4/6Ampholyte,40%,10ml163-11423713
Bio-Lyte4/6Ampholyte,40%,25ml163-11439287
Bio-Lyte5/7Ampholyte,40%,10ml163-11522984
Bio-Lyte5/7Ampholyte,40%,25ml163-11537540
Bio-Lyte6/8Ampholyte,40%,10ml163-11623569
Bio-Lyte6/8Ampholyte,40%,25ml163-11638187
Bio-Lyte7/9Ampholyte,40%,10ml163-11723479
Bio-Lyte8/10Ampholyte,20%,10ml163-11821912
Bio-Lyte5/8Ampholyte,40%,10ml163-11923275
Bio-Lyte5/8Ampholyte,40%,25ml163-11938114
(三)等电聚焦上样缓冲液(美国Bio-Rad公司),4℃冰箱保存
项目货号原价(元)
100×ReadyStripBuffer,forpH7-10IPGStrips,1ml163-2093744
Bio-Lyte3/10Ampholyte,100×,1ml163-2094396
100×ReadyStripBuffer,forpH6.8-8.3IPGStrips,1ml163-2095566
100×ReadyStripBuffer,forpH5.5-6.7IPGStrips,1ml163-2096774
100×ReadyStripBuffer,forpH4.7-5.9IPGStrips,1ml163-2097545
100×ReadyStripBuffer,forpH3.5-5.1IPGStrips,1ml163-2098592
1.2蛋白质定量试剂盒及其试剂
项目货号报价(元)
ProteinAssayKit
,bovineγ-globulinstandard500-0001919
ProteinAssayKit
,BSAstandard500-0002919
ProteinStandard
,bovineγ-globulin,1bottle500-0005429
ProteinAssayDyeReagentConcentrate,450ml500-0006690
ProteinStandard
,bovineserumalbumin,1bottle500-0007365
QuickStartProteinAssayKitⅠ,Bradford法500-0201934
DcProteinAssayKitⅠ,bovineγ-globulinstandard500-01112281
DcProteinAssayKitⅡ,BSAstandard500-01122281
RCDCProteinAssayKit
,γ-globulin,Lowry法500-01213183
RCDCProteinAssayKit
,BSAstandard,Lowry法500-01223183
1.3蛋白样品制备试剂盒及其试剂
项目货号原价(元)
Ready-Prep2-DCleanupKit163-21302533
ReadyPrepSequentialExtractionKit163-21002969
Tributylphosphine(TBP),200mM,0.6ml163-2101539
ReadyPrepSequentialExtractionKitReagent1,1vial163-21021116
ReadyPrepSequentialExtractionKitReagent2,1vial163-2103909
ReadyPrepSequentialExtractionKitReagent3,1vial163-2104646
ReadyPrep2-DStarterKit163-21053250
ReadyPrep2-DStarterKitRehydration/SampleBuffer163-2106331
ReadyPrepStarterKitEquilibrationBuffer
,withDTT163-2107508
ReadyPrepStarterKitEquilibrationBuffer
163-2108498
E.coliProteinSample,lyophilized,2.7mg163-2110446
ReadyPrepOverlayAgarose,50ml163-2111287
ProteoMinerProteinEnrichmentSmallCapacityKit163-30063699
ProteoMinerProteinEnrichmentLarge-CapacityKit163-20075618
AurumSerumProteinMiniKit732-67011513
AurumAffi-GelBlueMiniColumns732-67081873
ReadyPrepProteinExtractionKit(Cytoplasmic/Nuclear)163-20894919
ReadyPrepProteinExtractionKit(MembraneI)163-20884919
ReadyPrepProteinExtractionKit(MembraneII)163-20842986
ReadyPrepProteinExtractionKit(Signal)163-20872815
1.4化学试剂
项目货号原价(元)
尿素Urea,250g161-0730222
尿素Urea,1kg161-0731685
AG501-X8(D)MixedBedResin142-64252771
CHAPS,1g161-0460390
TritonX-100,500ml161-0407421
DTT(Dithiothreitol),1g161-0610291
DTT(Dithiothreitol),5g161-0611731
Tributylphosphine(TBP),200mM,0.6ml163-2101539
碘乙酰胺Iodoacetamide,30g163-21091429
溴酚蓝(BromophenolBlue),10g161-0404501
矿物油(MineralOil),500ml163-2129298
SDS,100g161-0301368
SDS,1kg161-03021991
Tris,500g161-0716813
Tris,1kg161-07191151
低熔点琼脂糖,25g161-31111360
甘氨酸,1kg161-0718713
甘氨酸,2kg161-07241671
丙烯酰胺(Acrylamide),99.9%,500g161-0101919
丙烯酰胺(Acrylamide),99.9%,2kg161-01033411
丙烯酰胺(Acrylamide),99.9%,1kg161-01071870
丙烯酰胺(Acrylamide),99.9%,5kg161-01088514
甲叉双丙烯酰胺(Bis),5g161-0200294
PDA(PiperazineDiacrylamide),10g161-02021902
PDA(PiperazineDiacrylamide),50g161-020317361
过硫酸氨(AmmoniumPersulfate),10g161-0700155
过硫酸氨(AmmoniumPersulfate),100g161-07541153
TEMED,5ml161-0800207
TEMED,50ml161-0801485
甘油Sigma
硫尿Sigma
1.5蛋白质标准品Marker
项目货号原价(元)
UnstainedSDS-PAGEStandards,highrange,200μl161-0303689
UnstainedSDS-PAGEStandards,lowrange,200μl161-0304689
UnstainedSDS-PAGEStandards,broadrange,200μl161-0317884
PolypeptideSDS-PAGEStandards,200μl161-0326692
PrestainedSDS-PAGEStandards,highrange,500μl161-0309689
PrestainedSDS-PAGEStandards,lowrange,500μl161-0305689
PrestainedSDS-PAGEStandards,broadrange,500μl161-0318800
PrecisionPlusStdUnstained,1ml161-03631168
PrecisionPlusStdDualColor,500ul161-03741080
2-DSDS-PAGEStandards,500μl161-03201457
IEFStandards,pIrange4.45-9.6,250μl161-03101621
1.6染色试剂
项目货号原价(元)
CoomassieBrilliantBlueR-250,10g161-0400413
CoomassieBrilliantBlueG-250,10g161-0406670
CoomassieBrilliantBlueR-250StainingSolutionsKit161-04351440
CoomassieBrilliantBlueR-250StainingSolutions,1L161-0436464
CoomassieBrilliantBlueR-250StainingSolutions,4×1L161-04371856
CoomassieBrilliantBlueR-250DestainingSolutions,1L161-0438488
CoomassieBrilliantBlueR-250DestainingSolutions,4×1L161-04391952
Bio-SafeCoomassieStain,1L161-0786878
Bio-SafeCoomassieStain,5L161-07873493
SYPRORubyProteinGelStain,200ml170-31262226
SYPRORubyProteinGelStain,1L170-31255725
SYPRORubyProteinGelStain,5L170-313816744
SilverStainKit161-04433933
SilverStainPlusKit(质谱兼容)161-04492854
1.7注意事项
1.双向电泳中所用的化学试剂纯度要高,至少为分析级,尽量选用进口试剂。
2.双向电泳中使用超纯水。
3.所有包含尿素的溶液加热温度不超过30℃,否则会发生蛋白氨甲酰化。
详细订货信息请致电:
-128
广州誉维生物科技仪器有限公司
Bio-Rad产品专员唐佩君
第二章SDS-PAGE聚丙烯酰胺凝胶电泳
2.1溶液的配制
30%聚丙烯酰胺贮液
丙烯酰胺150g
甲叉双丙烯酰胺4g
超纯水500ml
滤纸过滤后,棕色瓶4℃冰箱保存
1.5mol/LTris碱pH8.8
Tris碱90.75g
超纯水400ml
用1mol/LHCl调pH至8.8,加超纯水定容至500ml,4℃冰箱保存。
0.5mol/LTris碱pH6.8
Tris碱12g
超纯水120ml
用1mol/LHCl调pH至6.8,加超纯水定容至200ml。
4℃冰箱保存。
10%SDS
SDS10g
超纯水100ml
混匀后,室温保存。
10%Ap
Ap0.1g
超纯水1ml(用时加水溶解)
溶解后,4℃冰箱保存
10×电泳缓冲液
(1×=25mMTris,192mMglycine,0.1%SDS,pH8.3)
Tris碱30g
甘氨酸144g
SDS10g
超纯水1L混匀后,室温保存
2×SDS-PAGE上样缓冲液
0.5MpH6.8Tris碱2.0ml
10%SDS4.0ml
甘油2.0ml
1%溴酚蓝0.05ml
超纯水定容至10ml
DTT0.154g(用时现加)
2.2SDS-PAGE凝胶的配制
参见附录1
2.3操作方法
1.将玻璃板洗净、晾干、固定。
2.按照实验要求,配制不同浓度的分离胶。
将分离胶注入玻璃板夹层中,上部用超纯水、乙醇或水饱和正丁醇封面,保持胶面平整,待分离胶聚合后,配制浓缩胶。
3.配制好浓缩胶后,倒去分离胶表面的少量水分,然后灌入浓缩胶,插入点样梳。
4.将待分析鉴定的蛋白质样品,