1. Optional: "Renature" gel- this is thought to permit some refolding of proteins and may be important in finding epitope recognition of monoclonal antibodies. However its major advantage is probably that it removes the SDS from the gel which impedes transfer.
Soak for 30 min- 2 hrs in: 10 mM Tris pH 6.8 0.1 M DTT 4 M Urea 50 mM NaCl 2. Transfer 2-3 hrs- O/N at 350mA 3. Block in TBS containing 0.05- 0.1% Tween for 15 mins to O/N OR + 5% non-fat dryed milk for ECL at RT for 2hrs 4. Stain with Ponceau S for 5 mins 5. Dilute antibodies in blocking buffer. 1/500 for 125I blots for 2hrs RT 1/-5,000 for flag & myc and 1/3000 for 5172 for ECL O/N at 4C 6. 3 X 10 min washes in TBS-Tween. 7. Incubate in Rabbit anti-mouse 1-2 hr at RT (dilute 1/500) 8. 3 X 10 min washes in TBS-Tween.
9. Secondary antibody 1-2 hr at RT
(Use NEX-L- low specific activity 125I-protein A -diluted 2 x 105cpm/ml
(1uCi = 106 cpm)
dilute Goat anti mouse HRP-ab 1/4,000 for ECL
Add 1M NaCl and 0.5% Triton X-100 to the middle wash for 30 mins to 1 hr if background is a problem. ECL Incubate 1min @ RT in mix (1:1) of the two ECL solutions Dry out the excess liquid on Whatman paper Wrap the membrane in Saran wrap 10X Tranfer Buffer 30.27g/L Tris 8.8 -25mM 144.1g/L Glycine -192 mM ECL reagent: homegrown version Advantages: almost no cost; stable/reproducible, stored in frozen aliquots To 10 mls of 100 mM Tris pH 8.5 (RT), add 50 ls of luminol (warm to redissolve) 22 ls of coumaric acid (warm to redissolve) 3 ls of H2O2 (fresh < 6 months) Pour onto blot for 1 minute and process as normal (10 mls enough for 100 cm2) Stock luminol: 250 mM 3-aminopthalhydrazide (Fluka #09253); 266 mgs in 6 mls DMSO; store frozen in 60 ls aliquots. Stock coumaric acid: 90 mM coumaric acid (Sigma C9008): 38 mgs in 2.5 mls DMSO; store frozen in 25 l aliquots. Stripping blots: Most antibodies should be removed by heat and SDS: 100 mls -ME 100 mM 0.71 mls 14.1 M stock from bottle SDS 2% 20 mls 10% stock 62.5 mM Tris, pH 6.8 12.5 mls SDS-PAGE upper gel buffer Wash blot well with aq, including warm water, add stripping solution warmed to 50oC in closed container, incubate 30 mins, decant with large volume of running water, wash again with aq, and put into Blotto to reblot. Would recommend putting back into ECL to check efficiency of strip, and repeating the secondary control to check for background. Note foreground goes down with every strip, and background comes up.
To strip membrane after immunoblot - Heat Detergent-Manue
Stripping buffer: 100 mM ß-mercapto-ethanol, 2% SDS, 62.5 mM Tris-Cl pH 6.8
- ß-mercaptoethanol 176 µl 342 µl
- 20% SDS 2.5 ml 5 ml
- Tris-Cl pH 6.7 1.56 ml 3.125 ml
- to H2O 25 ml 50 ml
Incubate and agitate the membrane in stripping buffer for 30 min. at 50°C
Wash 6 X for 5 min. minimum each in 1X PBS - 0.1% Tween
Optional:
Incubate the membrane for 1 min. in the Western Blot Chemiluminescence Reagent
Expose the film for 1 min. to 1 hr to make sure that the original signal is removed
Wash the membrane again 4 X for 5 min. minimum each in 1X PBS - 0.1% Tween
The membrane is ready to reuse
Start at the blocking step
To strip membrane after immunoblot - Low pH-Manue
Stripping buffer: 25 mM glycine-HCl, pH 2, 1% SDS
- Glycine 1.876 g
- 20% SDS 50 ml
- to H2O 1 L
Incubate and agitate the membrane in stripping buffer for 30 min. at room temperature
Wash 3 X for 10 min. each in 1X PBS - 0.1% Tween
Optional:
Incubate the membrane for 1 min. in the Western Blot Chemiluminescence Reagent
Expose the film for 1 min. to 1 hr to make sure that the original signal is removed
Wash the membrane again 4 X for 5 min. minimum each in 1X PBS - 0.1% Tween
The membrane is ready to reuse
Start at the blocking step
Membrane: Protran (Schleicher and Schuell)
Rq: a PVDF membrane enables to sequence direcly from the membrane
Red ponceau S
Prepare a 2% (2 mg/ml) stock solution of Ponceau S dye in 30% (wt/v) trichloroacetic acid
Dilute the stock 10-fold in 1% acetic acid (final concentration of Ponceau S is 0.2%)
1X PBS - 0.1% Tween
- 5X PBS 200 ml
- Tween 20 1 ml
- to H2O 1 L
5% non-fat dry milk in 1X PBS - 0.1% Tween
- Non-fat dry milk 5 g
- to 1X PBS - 0.1% Tween 100 ml
Rq: this solution can be replaced by 3% BSA - 1X PBS - 0.2% Tween - sodiumazide (see Rodger)
Paint Pen Polymark: Natural Glow No. PM501, Dimensional Fabric and Graft
近日，MarketsandMarkets咨询公司发布了全球westernblotting市场的分析报告。据MarketsandMarkets预测，全球westernblotting的市场规模将从201......
近日，MarketsandMarkets咨询公司发布了全球westernblotting市场的分析报告。据MarketsandMarkets预测，全球westernblotting的市场规模将从201......
一、原理一个基因表达的最终产物是产生相应的蛋白，因此检测蛋白是测定基因表达的主要标志。 原理：WesternBlotting是将获得的蛋白质样品通过SDS-聚丙稀酰胺......
科学家经常通过蛋白质的表达及修饰情况来揭示对应样品的生理或病理状态。WesternBlot是进行蛋白检测的最经典技术。核酸检测对样品量的要求极低，因为DNA或RNA具备体外扩增的性质。而蛋白质检测只能......
Westernblotting是一个步骤繁多的实验，实验中的每一步都会对最后的结果产生影响，电泳过程是第一步，也是很重要的一步，好的电泳能让目的条带整齐，就像人工画的。以下是总结的一些影响实验结果的因......
Westernblot是检测蛋白质的一项经典技术，几十年来一直受到人们的欢迎。不过，令人尴尬的是，这也是一个相当耗时并且难以重现的过程，有时带来惊喜，有时则是惊吓。典型的Westernblot流程大家......
为了追踪Westernblot中产生的信息，研究人员大多需要使用成像系统。这些工具将蛋白分析的图像转换成数字文件。近年来，Westernblotting成像系统有了明显改进。在成像工具的帮助下，数据可......
常见问题及解答：1、两快玻璃板之间灌胶,胶为什么总是不平?1)你的玻璃洗干净没有？应该要洗得非常干净！2)过硫酸铵和TEMED的加量不合适，加量相对较多，凝胶凝固过快也会胶不平，最多按照分子克隆加倍3......
Westernblot，无疑是个老话题。然而，正是由于新产品的陆续问世，这颗老树才不断开出新花。两年前曾经介绍了全自动的Westernblot分析系统–SimpleWestern。这台名叫Simon的......
上海昆士兰生物科技发展有限公司是一家集研发、生产、销售于一体的高科技生物试剂公司。20年前本公司与中科院共同开发研制了辣根过氧化物酶（HRP）生产工艺，其纯度、精度达世界先进水平，现昆士兰生物开发的H......