Why do you bother about doing starch gels. About 10 years back, I performed several isozyme analyses for plant peroxidases, esterases, using native PAGE gels (no SDS anywhere). It worked out excellently. Try using PAGE gels to overcome your problems.
bye and best of luck
Jayakumar R. nair
Dept. of Cancer Genetics
Roswell park Cancer Institute
Buffalo NY 14263
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[mailto:[Only registered users see links. ].indiana.edu]On Behalf Of vijayendra
Sent: Wednesday, December 21, 2005 12:36 PM
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Subject: Starch gel
I am doing Starch gel electrophoresis in my lab for isozymis analysis
of plant peroxidase. But the problem i am facing is my gel doesnot get
I am using 0.3 M TBE buffer to prepare the gel.
Could anyone please suggest me what to do with this stuff?
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Different supplies of the soluble starch proved erratic in their gelling properties. As an alternative, soluble starch with the required characteristics has been made as follows : 300 g of potato starch (B.D.H.) are suspended in 600 ml of acetone-HCl (1 vol. of concentrated HCl to 100 vol. of reagent-grade acetone) at 38.5 °C. After standing for 45 min. without further mixing, 150 ml of aqueous 1 M sodium acetate is added to stop reaction. The starch is the filtered off on a Buchner funnel and washed thoroughly with dH2O. To remove any traces of acetate still remaining, the starch is resuspended in dH2O, allowed to stand overnight, washed again with dH2O on a Buchner funnel, dehydrated with acetone and finally dried thoroughly at 45-50 °C.
To try this
Running borate buffer : 0.25 M at pH 8.8 : dissolve 0.25 M H3BO3 and adjust pH with 1 M of NaOH and filing up 1 liter with dH2O
Gel borate buffer 0.025 M at pH 8.8 : dilute 1:10 running borate buffer 0.15 M at pH 8.8 with dH2O
Starch-gel : dissolve 14 g soluble starch in 100 ml di gel borate buffer 0.025 M at pH 8.8
Sample applicator : cut 5x7 mm Whatman n. 3 mm filter paper
Contact running buffer-gel : Whatman n. 3 mm filter paper whetting in running buffer for 10 min.
Potential delivered : 5.5 – 6.0 Volt/cm
Operative condition : room temperature
Running time : 3 hours
After electrophoresis, the gel is sliced in half horizontally and stained for peroxidases, using benzidine as the hydrogen donor . After 20 min., the stained gels are preserved by placing them in 50% methanol.