I'm trying to make a SDS-page of total proteins extracted from algae but I'm encountering two problems. First, when I depose my proteins in the wells the sample goes down and then it starts to move up the sides of the well. Second the result of my run looks like this: SDSpage vertical smear

so my bands are not visible and I have like a vertical smear on the sides of the lane.

The buffer used to extract proteins contains Tris, NaCl, EDTA DTT and Glycerol and my loading buffer is a 5x commercial dye with B-mer.

Thank you!

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    $\begingroup$ What did you expect? Single bands clearly visible? How much total protein did you load? $\endgroup$ – Chris Jul 27 '16 at 10:09
  • $\begingroup$ I loaded 10 ug of total protein extract and I expected to see at least some neat bands. Honestly I don't know if the "smear" effect is normal (but I've never seen that with bacterial protein extract), or if it can be related to the degradation of my sample or if it is just a problem in the migration. $\endgroup$ – Radioactivestar88 Jul 27 '16 at 20:15
  • $\begingroup$ 10ug is not very much - you could load more (20-30). Simply do this as a side by side comparison. Additionally: This is pretty much how I would expext a raw cell extract stained with coomassie to look like. There are hundreds of different proteins of different sizes in there, you would only see bands for really strongly expressed ones. Or for ones which you can enrich for. Otherwise I woud use Western blot to stain for specific proteins, which usually gives very nice bands even from raw extracts. $\endgroup$ – Chris Jul 27 '16 at 21:52
  • $\begingroup$ Indeed my goal is to perform a western blot but I'm not seeing a nice band where it should be, that's why I tried to compare my samples with a coomassie staining. :-) Ok, I'll try to load a bit more protein and I also tried to change the extraction buffer to solve the "moving up" problem. $\endgroup$ – Radioactivestar88 Jul 29 '16 at 7:42
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    $\begingroup$ You don't have to see a nice band on the coomassie stained gels to get nice bands in western blots. I do this all the time (doing whole cell extracts and use them for western), I can post the protocol I am using later today as an answer. $\endgroup$ – Chris Jul 29 '16 at 9:40

It's a guess, as I haven't run a algae sample on SDS-PAGE, but your migration problems might be due the algal polysaccharides that are present in your sample. Check if your protein purification protocol is specific for algal proteins and see if there is any step dealing with the algal polysaccharides.


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