HELP: Visualising Raw-Starch Binding Site Domain

C. SHAW (IND. MICROBIOLOGY) PG CSHAW at acadamh.ucd.ie
Fri Sep 13 11:55:57 EST 1996


Dear Netters,
    I have an alpha-amylase which possesses seperate sites for starch 
hydrolysis and binding to raw starch (and beta-Cyclodextrin).

   I have managed to cleave the enzyme using subtilisin to leave a smaller 
protein which still hydrolyses soluble starch but does not bind to or 
hydrolyse raw starch. However, I have difficulty visualising the 
cleaved-off protein fragment containing the binding site on a gel.
I have seen a band of about the right size on an SDS gel (using 
Pharmacia's PhastGel system) but under native conditions the protein 
cannot be detected. Gel filtration of the proteolysis products does 
not detect any new small protein peaks being eluted. 

  Ideally, what I need is to see the protein fragment in the gel 
binding to raw starch! Is this possible? I doubt that dyed starch, 
amylose azure, etc. would bind in sufficent quantities to be visible 
since silver staining is barely adequate. Is there such a thing as 
fluorescent starch or beta-CD available or some others means to label the 
binding substrate with significant sensitivity.

Any thoughts welcome,
Thanx in advance,

Cormac.

________________________________________________________________________
Cormac Shaw, BSc,                 |
Dept. of Industrial Microbiology, |  e-mail: cshaw at acadamh.ucd.ie
University College, Dublin 4,     |   phone: +353 1 706 1796
Ireland.                          |     fax: +353 1 706 1183
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"Under the most rigorously controlled conditions of pressure,
temperature, volume, humidity, nutrients and other variables,
the organism will do as it pleases."
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