zymogram of pectinase - activity staining of pectinase (Nov/17/2008 )

hi all,

i need ur help.
I am doing the pectinase purification
and now in the SDS step for knowing the molecular weight of my enzyme.

There are some question i would like to ask you:
1. i cant find the band of my pure enzyme in SDS gel, do u think is caused by the low protein content of my pectinase (it's about 0.05 micro gram/micro liter)?

2. for confirmation of which band that is my enzyme, i'll do activity staining. From the literature it's possible to overlaid the SDS gel with agarose contains pectin, and for visualization i can use CTAB and it'll appear clear zone as the hydrolysis zone, but in my case, although i use commercial pectinase, this CTAB couldnt work( i cant see clear zone). are there any suggestion?

thank you so much

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1) have you tried silver staining the gel to find your band?

2) you may need to renature your enzyme before exposing the gel to the pectin-agarose.

this can be done by soaking the gel in triton x-100 buffer.

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thanks for the response.

i havent tried using silver staining, because i dont have it.
now, i still try to concentrate again my pure enzyme with microconcentrator to get higher protein content.


for the zymogram, i already renatured with triton X-100 for 1 hour,
then washed with buffer 3x before overlaying in 1% agarose+ 0.1% pectin.
now i try with commercial pectinase to confirm the procedure and
i would try use iodine, maybe it can work (similar in pectinase screening agar),
anyone has another suggestion?

Thank you so much

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