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protein extraction from GEL


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5 replies to this topic

#1 Dr.Kiran

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Posted 08 July 2012 - 04:30 AM

Can anybody help me with protocol for protein extraction from sds polyacrylamide gel for raising hyper immune sera..the gel is used is of 15% strength from virus

#2 Adrian K

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Posted 08 July 2012 - 02:46 PM

Easy and fastest way: you can try electroelution. Can look for commercial kits such as pierce or merck.
There is a protocol on WILEY but I do not have access to it:
http://onlinelibrary...808s46/abstract

Another from SPRINGER (I think is free access)
http://www.springerl...36w023410u816u/

Also, you may consider:
http://onlinelibrary...CO;2-X/abstract
http://www.springerl...&page=4&locus=6


Hope this helps.
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#3 mdfenko

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Posted 09 July 2012 - 08:15 AM

you can excise the band, mash it and inject it (that's the way we did it). there is no need to remove the gel for inoculation.
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#4 biozweistein22

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Posted 09 July 2012 - 12:36 PM

First of all, mdfenko is right. The easiest way is to excise the band itself and mash it in PBS, but this will raise a huge immune response and may kill your animal because the gel is toxic, since you'll have to stain and destain it to see your band. If you do that, use coomassie and destain with 2-propanol instead of methanol as it is milder.

#5 mdfenko

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Posted 11 July 2012 - 05:43 AM

First of all, mdfenko is right. The easiest way is to excise the band itself and mash it in PBS, but this will raise a huge immune response and may kill your animal because the gel is toxic, since you'll have to stain and destain it to see your band. If you do that, use coomassie and destain with 2-propanol instead of methanol as it is milder.

the gel matrix is inert, not toxic (but seems to act as an adjuvant). coomassie is not toxic. acetic acid is not toxic. methanol could be toxic but will be in such low concentration after homogenizing the gel in buffer that it should not have any effect. plus, we always wash the gel in water or buffer to remove (or, at least, reduce) the destaining solution.
talent does what it can
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#6 biozweistein22

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Posted 11 July 2012 - 06:44 AM

Both coomassie and destain contain methanol, at least in our lab, that's why I said that the gel's staining and destaining makes it toxic. To avoid that, we wash the gel as you said using PBS before mashing it, and also use 2-propanol instead of methanol to make the stain-destain. In my experience it is much safer that way. Hope it works....




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