ProductDetails | |
---|---|
ProductType | Kit |
Properties | |
ApplicationSet | CompoundScreening |
OtherProductData | ClickhereforOriginalManufacturerProductDatasheet Ourproductdescriptionmaydifferslightlyfromtheoriginalmanufacturersproductdatasheet. |
Declaration | ManufacturedbyBioActs. |
ShippingandHandling | |
Shipping | BLUEICE |
ShortTermStorage | +4°C |
LongTermStorage | +4°C |
HandlingAdvice | Keepcoolanddry. Protectfromlight. |
Use/StABIlity | Stableforatleast1yearafterreceiptwhenstoredat+4°C. |
Documents | |
Manual | No |
MSDS | DownloadPDF |
ProductSpecificationSheet | |
Datasheet | DownloadPDF |
Flamma®749ProteinLabelingKitofBioActsisaproductdesignedtoeasilylabelantibodyorproteinwithvariousfluorescentsubstancesofFlamma®Fluors.Flamma®749ProteinLabelingKithasbeenpreparedtoproceed entireprocessfromlabelingreactiontorefinement.Flamma®749dyeinproductisareactivedyeinwhichvinylsulfonereactivegroupthateffectivelybondswithprimaryaminesofproteinisapplied,anditcancreateefficientprotein-dyecombination.Excitation/Emission(nm): 749/774nm.Extinctioncoefficient:220,000cm-1M-1.CF280:0.03.
ebiomall.com
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不知道发在这里合适不,实在是求助无门啊!版主手下留情。
百泰派克公司采用Thermo Fisher的Q ExactiveHF质谱平台,Orbitrap Fusion质谱平台,Orbitrap Fusion Lumos质谱平台结合Nano-LC,推出基于iTRAQ的定量蛋白组分析服务技术包裹,您只需要将您的实验目的告诉我们并将您的蛋白寄给我们,我们会负责项目后续所有事宜,包括蛋白酶切、肽段标价、肽段分离、质谱分析、质谱原始数据分析、生物信息学分析。
一、探针DNA标记方法
步骤 :
1.10ng~3ugDNA每管,双蒸水定量至总体积15ul
2.沸水水浴10分钟后迅速冰浴
3.加入 5号试剂 2ul , 6号试剂 2ul ,7号试剂 1ul
4.37度1小时~20小时
5.中止反应 加2ul 0.2M EDTA (pH 8.0) 和/或 65度 水浴 10分钟
二、标记效率检测
(一) 试剂配置
Solution Composition Preparation
Washing buffer 0.1 M Maleic acid, 0.15 M NaCl; pH 7.5 (20° C); 0.3% (v/v) Tween 20
Maleic acid buffer 0.1 M Maleic acid, 0.15 M NaCl; adjustwith NaOH (solid) to pH 7.5 (20° C)
Detection buffer 0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5 (20° C)
TE-buffer 10 mM Tris-HCl, 1 mM EDTA, pH 8.0
Blocking stock solution Dissolve Blocking reagent (bottle 10) 10% (w/v) in Maleic
(10 × conc.) acid buffer underconstantly stirring on a heating block(65°C) or heat in a microwave oven,autoclave. The solution remains opaque
Blocking solution Prepare a 1 × working solution by dilutingthe 10 × Blocking solution 1:10in Maleic acid buffer.
Antibody solution Centrifuge Anti-Digoxigenin-AP(vial 8) for 5 min at 10 000 rpm in theoriginal vial prior to each use, and pipet the necessary amount carefully from thesurface. Dilute Anti-
Digoxigenin-AP 1: 5 000 (150 mU/ml) in Blocking solution.
Colorsubstrate Add 40 _l of NBT/BCIP (vial 9) to 2 ml of Detection buffer.
solution Note: Store protected from light!
(二)对照的标记DNA(4号试剂)系列稀释
(三)步骤
1、取以上制备的管2~9各1ul,以及自己标记的探针1ul,点到一小片尼龙膜
2、通过紫外线或者120度半小时使核酸交连到膜上
3、膜置塑料盒中加Maleic acid buffer 20ml ,15~25度轻摇孵育2分钟
4、10ml Blocking solution 孵育30分钟
5、10ml Antibody solution 孵育30分钟
6、10ml Washing buffer 洗2次,每次15分钟
7、10ml Detection buffer中平衡2~5分钟
8、膜在2ml新鲜配制的 Colorsubstrate solution 中避光孵育。显色期间避免摇动
9、中止反应 用TE-buffer或者双蒸水洗5分钟
蛋白酶K预处理
三、样品检测与杂交
(一) 步骤
1、 稀释供试品及阳性对照DNA,点膜
2、 通过紫外线或者120度半小时使核酸交连到膜上
3、 将标记好的探针稀释到约25ng/ml, 煮沸5分钟后迅速冰浴
4、膜放入预热好的预杂交液(3.5ml/100cm2)中,充分混匀,避免起泡
5、倒掉预杂交液,加入杂交液及已变性的探针。杂交温度下至少孵育6小时
四、洗膜
1、2 × SSC, 0.1% SDS , 15-25° C,洗膜2次,每次5分钟。
2、0.5× SSC, 0.1% SDS ,预热至65~68° C,洗膜2次,每次5分钟
五、结果检测
(一) 试剂配制
Solution Composition Preparation
Washing buffer 0.1 M Maleic acid, 0.15 M NaCl; pH 7.5 (20° C); 0.3% (v/v) Tween 20
Maleic acid buffer 0.1 M Maleic acid, 0.15 M NaCl; adjustwith NaOH (solid) to pH 7.5 (20° C)
Detection buffer 0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5 (20° C)
TE-buffer 10 mM Tris-HCl, 1 mM EDTA, pH 8.0
Blocking stock solution Dissolve Blocking reagent (bottle 10) 10% (w/v) in Maleic
(10 × conc.) acid buffer underconstantly stirring on a heating block(65°C) or heat in a microwave oven,autoclave. The solution remains opaque
Blocking solution Prepare a 1 × working solution by dilutingthe 10 × Blocking solution1:10in Maleic acid buffer.
Antibody solution Centrifuge Anti-Digoxigenin-AP(vial 8) for 5 min at 10 000 rpm in theoriginal vial prior to each use, and pipet the necessary amount carefully from thesurface. Dilute Anti-
Digoxigenin-AP 1: 5 000 (150 mU/ml) in Blocking solution.
Colorsubstrate Add 40 _l of NBT/BCIP (vial 9) to 2 ml of Detection buffer.
solution Note: Store protected from light!
(二) 步骤
1、Washing buffer洗膜1~5分钟
2、100ml Blocking solution 孵育30分钟
3、20ml Antibody solution 孵育30分钟
4、100ml Washing buffer 洗2次,每次15分钟
5、20ml Detection buffer中平衡2~5分钟
6、膜在10ml新鲜配制的 Colorsubstrate solution 中避光孵育。显色期间摇动
7、中止反应 用TE-buffer或者双蒸水50ml洗5分钟展开
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