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I am currently working on SDS-PAGE technique having 20% acrylamide concentration for hydrolyzed BSA protein. Here I attached a gel photo for trypsin hydrolyzed BSA. I don't know , is it a good result and how do I interpreted this result?

20% sds page

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  • $\begingroup$ A 20% SDS gel? How big are the fragments you are expecting? The concentration seems rather high for me. $\endgroup$ – Chris Sep 23 '14 at 9:31
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    $\begingroup$ why do you need to know (is this for a report, assignment, or research?) it may affect the type of answer, and some more detail of method might be needed & what you are testing $\endgroup$ – rg255 Sep 23 '14 at 10:11
  • $\begingroup$ I am doing research on drug delivery. This my initial task to check hydrolysed protein on PAGE. protein hydrolyzed by trypsin chmotrypsin and pepsin in our stomach. $\endgroup$ – kt123 Sep 23 '14 at 10:30
  • $\begingroup$ her I am using 20% because on 15% I got band at bottom of GEL, nearer to edge. $\endgroup$ – kt123 Sep 23 '14 at 10:33
  • $\begingroup$ It looks like your trypsin works, at least. Is there something else you were expecting? I don't do very much SDS-PAGE(so take with a grain of salt), but I would lower the thickness of your gel and let the large blurry bands run off. The BSA and hydrolyzed protein fragments are those thin lines at the top. $\endgroup$ – Resonating Sep 23 '14 at 11:06
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I'm going to treat this as a partial homework question but provide some guidance as to how you can potentially address your question and have solid theory to back it up. Chymotrypsin preferentially cleaves peptide amide bonds where the carboxyl side of the amide bond (the P1 position) is a large hydrophobic amino acid (tyrosine, tryptophan, and phenylalanine). So one solution which is often used by experimental biologists (such as myself) performing mass spectrometry (MS) is to put their protein sequence (in this case BSA) in an algorithm such as PeptideCutter (from ExPASy), which performs a theoretical digest of the protein sequence having chosen the desired enzyme or combination of enzymes) and see what peptide fragments are expected to be produced and work out their length/molecular weight and compare that to your experimental data.

Having had an initial look, I noticed that your protein marker/ladder is missing, which is quite important in figuring out the peptide sizes produced! Also what is 1-8 lanes? are they different concentration of enzymes? if so then it looks ok to me since the higher the enzyme concentration from left to right, the higher the amount of digest products you have. If the peptide sizes obtained are not the ones expected then perhaps increase or decrease the digestion time since you could have a problem due to either incomplete digest or unspecific digestion, depending. I guess since BSA MW is ~ 66kDa your gel concentration is appropriate since you have a problem with the 15% gel as the products are too small and too abundant to resolve.

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  • $\begingroup$ I disagree on the gel percentage. I did run a lot of gels wit proteins in the 50-70 kDa range and I used 7 or 8% gels for that. The density here is ways too high, the undigested protein doesn't even enter the resolving gel, it stays at the border between stacking and resolving gel. At this position I would expect proteins around 100-120 kDa. What didn't work in the 15% gel? Can you use a marker? $\endgroup$ – Chris Sep 24 '14 at 6:50
  • $\begingroup$ I agree that 20% gel is quite high but OP in the comment section says using 15% gel fails to resolve the digested bands (which are many from the pic) as all collect at the bottom although I will be surprised if the large digested band also disappears. Hence the resolving gel (i assume) was used at 20%, which gave better results. It is important however to use a protein ladder just to see how proteins migrate as it is hard to tell at the moment what we are looking at. I personally use gradient gels 4-12% pre-made but thats ideal for proteins between 60-220kDa as I get an excellent resolution. $\endgroup$ – Bez Sep 24 '14 at 8:19
  • $\begingroup$ We don't know why the 15% gel failed as we never heard anything else about it. I see only one band per lane, the rest are the dyes used in the buffers. Additionally I wonder how he handled and stained a 20% gel, as these should be really brittle. I do 15% gels for oligo analysis from time to time and these are already hard to handle. $\endgroup$ – Chris Sep 24 '14 at 8:45
  • $\begingroup$ I expect most of BSA digestion products are to be small and various in size (smear like), which is what he is seeing, except that one band since the enzyme does not digest too specifically. Hence I do not think the smears he is seeing is non-washed stains since in between the lanes the SDS-PAGE gel is clear so he has got most of the unbound coomassie stain off although I do not know of his wash procedure. Oligos give a similar result to the above but I did not know you can run them in a SDS-PAGE as I just come across them in my agarose gel work. $\endgroup$ – Bez Sep 24 '14 at 9:47
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    $\begingroup$ It would however be nice for the OP to do an overnight wash (with a slightly modified wash buffer composition) to see if he/she gets a better resolution. Almost 100% of the times overnight washes work best and show bands, which were unresolved for me! but @Chris I agree about the general thrust of your argument specially the gel brittleness issue! $\endgroup$ – Bez Sep 24 '14 at 10:03

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