On Thu, 20 Aug 1998 05:39:12, delgadoi at PILOT.MSU.EDU (Ivan J Delgado Orlic)
wrote:
> Dear All,
> I am having problems with 1-D SDS-PAGE. After a lot of
> troubleshooting, I
> have concluded that there may be something wrong with my Arabidopsis protein
> samples (since other protein samples run fine into the same gel, including
> older Arabidopsis protein samples, but every single sample, old and new, tends
> to show protein bands only larger than about 25 kD). A copy of a Ponceau
> stained gel showing the "smiling" effect I see starting at around 29 kD (the
> greater the protein concentration, the more pronounced the smiling is) and the
> lack of polypeptides smaller than 25 kD can be seen here: http://www.msu.edu/~delgadoi/ponceau.html> The extracts are from Arabidopsis seedlings ground in a mortar and pestle,
> spun at 2,000g twice (each time the supernatant was transferred to a clean
> tube) and then desalted using a Centricon (5 kD cutoff) filter before
> resuspending in PBS (plus 0.1% Triton X-100). The buffers I have use for
> extraction include Laemmli buffer and a Tris+sucrose based buffer.
> Since I have never seen this before, any help would be greatly
> appreciated. I am running out of things to try (the acrylamide, Running buffer
> and the other reagents are nearly all from lab stocks that show consistent
> good results -they work!- so I believe the problem is with the protein sample,
> but what?).
> Thank you for your time,
> Ivan
>>> Ivan J Delgado Orlic Lab: 517-353-3519
> MSU-DOE Plant Research Laboratory delgadoi at pilot.msu.edu> East Lansing, MI 48824-1312 http://www.msu.edu/user/delgadoi
Thanks for the photo -- great idea! Looks to me like your samples have a ton of
salt in them. That would account for the smearing, smiling, and retardation of
low-mol wt polypeptides while the larger species aren't so much affected. The
salt front can be spread out a lot, and would interfere with lower-mol wt
species or make them run together. I think you'd want at most 50 mM monovalent
salt, and preferably <5 mM divalent cation.
Can you desalt by dialysis? On a micro-scale, use a 3500-MWCO membrane
stretched over a 'decapped' eppitube & held on with a ~2 mm ring of rubber
tubing or a rubber band, then floated upside down on buffer.
Sucrose in your buffer might well interfere with the centrifugal dialysis your
trying. I'd also recommend a more conventional extraction buffer unless, as I
infer, you're running a non-denaturing gel. Have a look at the Methods in Plant
Molecular Biology series book or other protein methods guide. For denaturing
gels, an extraction buffer with Tris, SDS, DTT, and PVPP should be fine -- I'd
include PVPP to chelate polyphenolics/tannins unless you know it's not needed.
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