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I want to separate 1600 Da peptide on SDS PAGE, on 20% GEL I didn't get any band, How can I separate on PAGE? Can I use 25% SDS PAGE?

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  • $\begingroup$ Didn't you get any bands because you sample run off the gel or because of other problems? $\endgroup$ – Chris Nov 11 '14 at 7:35
  • $\begingroup$ @Chris,yes,because of sample run off the Gel. $\endgroup$ – kt123 Nov 11 '14 at 8:21
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    $\begingroup$ Well you could turn down the power or reduce the time. What are you trying to separate it from? $\endgroup$ – canadianer Nov 11 '14 at 9:42
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    $\begingroup$ Then the answer is simple: Don't run the gel for too long. Sounds simple, but is not necessarily so. Your samples either run with or even before the blue line of the bromphenol blue. o if you run your gel only until 2/3, this should work. $\endgroup$ – Chris Nov 11 '14 at 9:58
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    $\begingroup$ @inf3rno I doubt it. 20% is already hard to handle as the high percentage gel get very brittle. $\endgroup$ – Chris Nov 11 '14 at 13:30
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                                                   enter image description here

Image is of an 18% Tris-tricine small-peptide SDS-polyacrylamide gel stained with coomassie blue dye [source].

Your peptide is 1.6 kDa; it would not run off in 20% gel if you don't run it for a long time. Also, try to prevent heating up of the gel (you can run it in a cold room or in front of an A/C vent). However, it is better to use other techniques such as chromatography. You can also try dialysis if other unwanted peptides are much larger.

Protocol for Tris-Tricine SDS-PAGE.

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If band is below the dye front it might not stain very well. Tricine gels are best for such small proteins, and you might might just run until dye front is only half way down the gel. It is also possible that your protein does not stain with conventional coomassie-based stains. Maybe try a silver stain.

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