English is not my mother tongue, if there are any unclear expression, please ask me directly.
Some information of my experiment:
15% SDS-PAGE, no stacking gel, run at 100v.
Lanes from left to right: marker(10~180kb), BSA(0.125mg/ml), sample1(0.65mg/ml), sample2(1.28mg/ml), sample3(1.19mg/ml), sample4(about2.3ml/ml), sample5(0.128mg/ml), and marker. The content of protein is determined by Bradford. Loading amount is 10μl, marker is 6μl.
sample1~4 are extracted from traditional Chinese Medicine of animal sources, extracted with 0.9%NaCl, and keep in -80℃.
Before run sds-page, mix 40μl sample and 10μl 5x loading buffer uniform, Heat in boiling water for 5 to 10 minutes. After running, incubate 1~2 hours with distilled water, until loading buffer disappear, and then incubate 1~2 hours with fast stainning Coomassie blue dye.
There are some questions in my page gel.
1. Why my marker go to another lane?
2. Why all the lanes are bule, and the bands not clearly?
3. Why the color is so light?
I dont know is 0.9%NaCl effect sds-page or not, and is there any questions about my experiment process?
Looking forward to your answer, thank you.
你好，一条一条回答你的问题：
1. Why my marker go to another lane?
加marker的时候，可能有一小部分marker漂移到旁边的lane去了。下次加的时候，把枪头伸得更深一些；同时做胶的时候，要注意孔道是否完好封闭。
2. Why all the lanes are blue, and the bands not clearly?
从胶图来看，你的蛋白上的有点多。可以稀释一半再上。不过其实可以看到几个比较浓的条带了。下次上的时候可以稀释，并且跑的时间再久一些，有助分离。染色的时间可以久一点，过夜脱色，直到胶的背景变成无色的。
3.Why the color is so light?
染色时间太短了。你也可以再脱色后，再加考马斯染一次，可以加重条带。
谢谢@Zhongtian Yu 的回答，我在制胶的时候加样孔里有胶，所以上样的时候好像确实有些飘出来了，下次会注意加样的问题。
下次我再把浓度调小一点上样，同时为了确定是不是样品中NaCl的问题，我会再换一些缓冲液试试。
1. When loading protein in gel, make sure the loading volume is not higher than the volume of the wells. Also, make sure you use a pipette tip with a capillary end for precision loading, minimizing the risk of the sample drifting to the next wells.
2. The running time was not enough for clear separation.
3. Your staining time might be too short.
- Badges
- Science method