Hi everyone,
We see two transparent lines (like the one separating the stacking and running gel) during the running and we also note that 25 and 20 MW band get stack in the upper transparent band. When we do the Coomassie staining we get a blue bar that is between the transparent lines we see during the running (Image attached).
We use this protein marker (DUAL COLOR) http://www.bio-rad.com/es-ar/product/prestained-protein-standards
What is the reason for lines in separating gel?. Available from: https://www.researchgate.net/post/What_is_the_reason_for_lines_in_separating_gel [accessed Jul 31, 2017].
I dont understand your question clearly but I think it is for your loading dye, so if your band is upper the line this line in not be important for you,if not,you can increase of your running time or increase destaining time.
I hope be useful.
The problem is that the 25 and 20 bands stack when these two transparent lines appear and then we really don't know if our protein is the right size or not.
As I said you can increase time of running and destaining. Also you can use 18% gel because your MW is low. Another solution that I think be helpful is use tricine gel or stain with silver.
The staining is not the problem. The problem is, at first all the bands are separating well, but when the transparent lines appear only the 20 and 25 kDa get stacked and continue stacking even if the gel runs more time, even though the 15, 10 and 5 bands don't get stacked.
55. A thin layer of Coomassie Blue on gel surface after destaining
a) Can easily be removed by a quick rinse in 50% methanol or by gently swabbing the gel
surface with destain-soaked tissue paper.
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