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HELP ME!! i need a simple protocol


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#1 ikwana

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Posted 24 January 2011 - 06:20 PM

i need to purify n isolate allinase enzyme from garlic... BUT! from all jurnal that i've read, a lot of chemical are not available in my campus..

for example:-pefabloc,PLP,PEG-800, methyl-alpha-D-mannoside, HiLoad 26/60 superdex 200 prep grade,alpha-globulin, racemic alliin,lactate dehydrogenase, NADH

i really need any other procedure that only use simple chemical which all lab must have.. i can't order all the chemical above because the shipping time takes about 6 month n i have to finish this project this june..

i don't know what to read anymore.. help me plzz.. this is my final year project...

#2 K.B.

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Posted 25 January 2011 - 02:40 AM

Three major question come to my mind:
1. What purity you need to get? Is this project have to end with very high (eg. 90-99%) purity or is it just an attempt to purify to test your skills?
2. What equipment, materials, and methods do you have access to?
- chromatography - what systems (gravity flow, low pressure, SPE, FPLC, HPLC?) and columns (size exclusion, ion exchange, reversed phase?) do you have?
- electrophoresis?
- ultrafiltration and/or dialysis?
- lyophilization?
- centrifugation?
3. Do you have established protocol to test for this enzyme activity?

#3 ikwana

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Posted 25 January 2011 - 06:28 AM

Three major question come to my mind:
1. What purity you need to get? Is this project have to end with very high (eg. 90-99%) purity or is it just an attempt to purify to test your skills?
2. What equipment, materials, and methods do you have access to?
- chromatography - what systems (gravity flow, low pressure, SPE, FPLC, HPLC?) and columns (size exclusion, ion exchange, reversed phase?) do you have?
- electrophoresis?
- ultrafiltration and/or dialysis?
- lyophilization?
- centrifugation?
3. Do you have established protocol to test for this enzyme activity?


1. the purification is for determination of its antioxidant activity. i dont know the percentage
2. i think the system is gravity flow, and using size exclusion column. i only have polyacrylmide gel but i dont have the HiLoad 26/60 superdex 200 prep grade
i'm going to blend my sample and seive it and undergo centrifugation before separate in the column

3. i do refer to one journal.. i refer all the procedure from this journal..
u may download the attachment to read the journal..

#4 K.B.

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Posted 25 January 2011 - 07:37 AM

First of all - since you need an active enzyme, not just inert protein, you MUST have some way to measure enzyme activity. It is not only to know the final result but also to monitor purification steps.

You have basic chromatography setup - that's good. Polyacrylamide size exclusion gel is most probably Bio-Gel P.

as for the methods from the paper you provided:
extraction buffer - I assume you have glycerol, salts and sodium azide, right? Phosphate or Tris should work as good as Hepes. Pefabloc (protease inhibitor) is quite important but it may work without it if you work quickly and at low temperatures. PLP - pyridoxal phosphate - is said in the article to be enzyme cofactor, so it most probably won't have any activity without it...
PEG-8000 - it is used for protein precipitation and there are other (cheap and simple) methods of protein fractionation that can be used e.g. ammonium sulphate precipitation, acetone precipitation, methanol or ethanol precipitation.

I would attempt to do this like that:
1. prepare extraction buffer as similar as possible to one from the article
2. blend garlic, extract for 15-60 minutes at +4 deg.C, and centrifuge (at least 10000*g for 15-60 minutes) [take sample of supernatant for analysis!]
3. perform ammonium sulphate precipitation stepwise from 0% to 100% saturation in 10% increments.
4. a) perform SDS-PAGE on samples from step 2 and 3, look for allinase band (approx. 51.5 kDa) (you can also make separate gel and stain for glycoproteins)
or
B) perform enzyme activity measurement on samples from step 2 and 3
(it would be best to perform both)
5. pool samples with high allinase activity / correct band and perform gel filtration
6. repeat step 4 with fractions from gel filtratio

[edit: to remove emoticon that got automatically inserted where it didn't supposed to be]

Edited by K.B., 25 January 2011 - 07:40 AM.





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