Help with SDS-PAGE of Arabidopsis samples

Ivan J Delgado Orlic delgadoi at PILOT.MSU.EDU
Thu Aug 20 00:39:12 EST 1998


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



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