protein extraction from GEL - (Jul/08/2012 )
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
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.wiley.com/doi/10.1002/0471142735.im0808s46/abstract
Another from SPRINGER (I think is free access)
http://www.springerlink.com/content/r036w023410u816u/
Also, you may consider:
http://onlinelibrary.wiley.com/doi/10.1002/1522-2683(200102)22:3%3C394::AID-ELPS394%3E3.0.CO;2-X/abstract
http://www.springerlink.com/content/m048g2437xk8t1q1/#section=85346&page=4&locus=6
Hope this helps.
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.
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.
biozweistein22 on Mon Jul 9 20:36:26 2012 said:
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.
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....