I see these dark smears on the top of the gel. I am worried about this because I need to quantify the bands. Some of the gel has even weaker bands than these, so I assume a large quantity of the protein went to the smears.
Could anyone tell me what those smears are? Is there any way to avoid this?
See also:
https://www.researchgate.net/post/Does_anyone_know_why_there_is_a_partial_smear_in_this_SDS_PAGE_picture_attached
https://www.researchgate.net/post/Can_anybody_please_suggest_what_is_the_reasons_of_having_smear_instead_of_separated_bands_in_SDS-PAGE
https://www.researchgate.net/post/Why_is_smear_formed_when_during_SDS_PAGE
Hi, how about you first quantify the protein with Bradford assay before you load on gel?
If your protein was degraded those smears should be moving downwards not upwards. I normally load 15ug of total protein/well.
Thanks for the suggestion, the problem is probably caused by the high salt condition in the running buffer or I have overloaded the protein.
This time when I run the gel, I intentionally add a little 10X running buffer into the previous 1X running buffer, because I have had problem of "no load detection" on the electrophoresis machine when I used 1X buffer, and I overcame the problem with adding more 10X buffer into it. Maybe this time when I add the 10X buffer, it caused this smear problem. Next time I will use very fresh buffer anyway.
I have a question, how do you quantify the bands based on a ladder e.g., 6x His ladder? I need to know what the concentration of my protein into 10 mL crude extract from cell lysis is and how much it was into the growing medium of 50 mL.
Can you help, as I am not confident with the way I have told to perform? thanks
Hi Nahed, it depends on what dye you used to stain your gel or membrane. Usually commassie blue detection limit is around 0.3ug-1ug and silver stain detection limit is from 0.5ng-5ng, western blot with HRP antibody staining, usually 0.1ng. So according to your band, you can approximately estimate the amount of protein in your lysate. I am not sure if you can use the marker to estimate the amount of your protein, if your marker is pre-stained, it will be hard.
I think that you might overloaded protein and also maybe used too high voltage to run the gel. Check also if reagents U are using for electrophoresis buffer are fresh because in my case it apperaed that SDS was too old. With the new one the quality of bands was much better.
your distilled water does'nt suitable. Therefore, gels are smiling face.
Hi,
1. check u run the gel at CC mode of 10-15mA till dye reaches the separating gel and then at CV mode of 10-15V till the dye reaches the bottom.
2.you try using 4X buffer
I would try loading less protein, overloading the gel also causes smears.
At what temperature you did boil/heat your samples? Try to reduce the time or the temperature. Too much protein may have a similar effect on the SDS-PAGE. Please try to load less protein and be sure that the loading dye is fresh.
Good Luck,
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