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troubleshooting for zymograme

enzyme zymograme Malate dehydrogenast

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3 replies to this topic

#1 piyushdravid

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Posted 25 April 2013 - 11:07 PM

Well, I was trying to purify malate dehydrogenase from parasite. The attached file shows the zymograme developed by incubating the native gel in the substrate Malate, NAD, PMS and NBT. The trouble is that i was getting less smearing in crude lysate (Lane2) and high smearing in partially purified fraction of lysate (lane 4). The enzyme has been partially purified using ammonium sulphate precepitation. Now i would like to reduce the smearing that was observed in partially purified fractions. Condition of native page-
Stacking- 0.5M Tris pH6.8 (4%)
Separating- 1.5M Tris pH 8.8 (7.6%)
running time- 2Hrs at 4 degree c

Thanks!!

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#2 mdfenko

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Posted 26 April 2013 - 04:19 AM

a couple of things to consider.

have you completely solubilized the ammonium sulfate fraction? dialyzed away residual ammonium sulfate?

you may want to decrease the loading of the partially purified sample.
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#3 piyushdravid

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Posted 26 April 2013 - 05:17 AM

a couple of things to consider.

have you completely solubilized the ammonium sulfate fraction? dialyzed away residual ammonium sulfate?

you may want to decrease the loading of the partially purified sample.

i have tested the partially purified fractions for ammonium sulphate using saturated solution of barium chloride. As no white precipitate observed, i am assuming that the fractions doesn't contain residual ammonium sulphate. I have also tried low amount of fractions on native page.
Is it possible that the enzyme has degraded during partial purification process or during the running of native page that's why smearing is observed in zymograme?

#4 mdfenko

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Posted 26 April 2013 - 08:10 AM

some of the smearing may be caused by degradation but anything above the band of interest would be aggregates.

after solubilizing (and dialyzing) the ammonium sulfate pellet you should clarify the solution to remove any particulates and, hopefully, aggregates.
talent does what it can
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