| 查看: 709 | 回复: 0 | |||
willer金虫 (正式写手)
|
[交流]
【分享】Western blot 常见问题及解决方案
|
|
转自http://www.molecularstation.com/ ... western-blots/#weak Troubleshooting Western Blots Problem: Spots on Film (missing bands) : - Poor Transfer due to air bubbles during transfer to membrane - dirty film when adding to the developer - developer rolls are dirty Solution to spots on film: - use a pasteur pipette as a roller. roll the membrane and gel before transfer to remove bubbles. keep membrane and materials wet. - keep film clean before adding to the developer Troubleshooting Western Blots Problem: Too Many Bands in Western Blot : - usually due to many non-specific bands - poor primary antibody quality or old antibody - not enough blocking - proteolytic cleavage of antigen - all additional bands are lower MW than your protein, ( ie your protein is being detected but was degraded) Solutions to too many bands on western blot: - purchase another antibody or replace with a fresh antibody stock - block with 5% dry non-fat milk or BSA prior to adding primary. If you are already blocking, consider blocking during primary and secondary antibody incubations and also increasing blocking milk or BSA concentration. - keep everything on ice, use fresh samples, store in - 80C, and use protease inhibitors such as PMSF Troubleshooting Western Blots Problem: High background; low signal to noise ratio on Western Blot - black or every dark film; film was exposed too long - blocking was insufficient ; non-specific binding of primary antibody and secondary antibodies were not washed completely - washing was insufficient - film glows in dark! - too high secondary or primary dilutions Solutions to Dark Film: - expose for less time, decrease exposure time - increase blocking duration of non-fat dry milk 5% incubation or increase the concentration. - also you can try blocking with whole serum of the host animal with (or before) the secondary antibody - increase washing times and volumes to help remove any non-specific signal due to weak antibody binding. - using a stronger detergent to wash - Instead of TBST (Tween-20), use stronger detergents such as NP-40 or SDS which may provide a more stringent wash and reduce background (do this only if you band is strong! - washing also removes some your band of interest!) - very high secondary antibody (or even primary) concentration - perform optimization experiments to determine proper antibody dilutions. dilute the antibodies further. Troubleshooting Western Blots Problem: Low Signal or Weak Signal - due to not enough protein loaded on the gel - primary antibody is old or not good - phospho-proteins usually need overnight primary antibody incubation - not enough secondary antibody - over-blocking - developing reagents are bad / you made a mistake when preparing them Solutions to Low Signal or Weak Signal: - load more protein sample onto gel - try fresh primary antibody - incubate primary antibody overnite for phosphoproteins at 4 degrees C (cold room shaker) - increase concentration of protein (lyse samples in less lysis buffer) - decrease concentration of blocking agent - check the developing reagents; prepare fresh ECL and re-ECL the membrane - increase the concentration or the length of primary antibody incubation period - increase concentration or the length of secondary antibody incubation period Fuzzy Bands, Bands Smeared, Bands not Sharp - bands smeared due to hot gel - bands fuzzy due to high voltage Solutions to Fuzzy bands and Un-sharp Bands: - decrease voltage or current, run in cold room, and prepare new running buffer. ( heat causes the gel to lose its rigidity and its resolving power) - pre-soak transfer membrane in the appropriate transfer solution (determ |
回复此楼» 猜你喜欢
生物质炭与活化剂氢氧化钾混合注意事项
已经有0人回复
-
科研人必读：新一代 KRAS G12C 抑制剂 Olomorasib 的研究进展
已经有0人回复
-
化学工程及工业化学论文润色/翻译怎么收费?
已经有121人回复
-
BT-11（Omilancor）研究化合物全解析：从分子结构到实验应用要点
已经有0人回复
-
富马酸贝达喹啉：二芳基喹啉类工具化合物的化学特征
已经有0人回复
-
BT投稿with editor状态快两周了，正常吗
已经有25人回复
-
Silevertinib（BDTX-1535）结构拆解：炔基双环片段为何成为合成瓶颈
已经有0人回复
-
RO4929097 解析：双苯并氮杂䓬骨架与 Notch 通路实验设计要点
已经有0人回复
-
分子量 1157 的 PROTAC 怎么配不析出：SGK3-PROTAC1 的溶解、分装与图谱复核
已经有0人回复
-
logP 6.1 的羧酸怎么配母液：GSK 650394 的溶解窗口与稀释顺序
已经有0人回复
-
从"水不溶"到 71 mg/mL：雷特格韦的 pH 依赖溶解与螯合色谱边界
已经有0人回复
找到一些相关的精华帖子，希望有用哦~
关于western blot的小问题
已经有12人回复
- western blot时marker问题求助
已经有18人回复
- Western blot中出现的问题-求高手指点
已经有17人回复
- Western Blot电泳蛋白质外漏问题求助
已经有22人回复
- 新手求助western blot
已经有22人回复
- 【分享】新手上路 Western blot问题集合
已经有6人回复
- 【求助/交流】western blot
已经有35人回复
- 【求助/交流】有关western-blot的一些问题
已经有18人回复
- 原创首发：Tips for Western Blot-一些小技巧而已
已经有13人回复
点击这里搜索更多相关资源科研从小木虫开始，人人为我，我为人人
