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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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eluding proteins fom PAGE gel
Posted by: kavitha (IP Hidden, Unregistered user, )
Date: December 21, 2004 07:20AM
I want to elude 24KD protein from native PAGE gel without loss of activity. If I use dialysis bag for this purpose. what will happen to the Coomassie blue stain?Will it remain within the bag or go into the solution? Is there any cheaper alternative method for eluding the proteins?
Re: eluding proteins fom PAGE gel
Posted by: huangz123 (IP Hidden, Junior member, 19)
Date: December 21, 2004 09:15AM
I don't think the coomassie dye will come out by simply putting the gel slice in dialysis buffer. You should try to electroelute your band from the gel. Or by conventional way to destain the gel slice it should be sufficient for assays like MS.
zhong
Re: eluding proteins fom PAGE gel
Posted by: mamuller (IP Hidden, Junior member, 10)
Date: January 31, 2005 04:45AM
To recover active and healthy protein you need to use
a light staining solution, commassie is a protein hammer, which limit your protein recovery. use SB010 staining solution 10ng sensitivity, staining during process & and once you have location of protein can removed with 1 wash distilled water. The kindest protein healthy efficent recovery system is electro elution with GeBAflex-tube. See www.geba.org Protein from gel extraction Maurice
Re: eluding proteins fom PAGE gel
Posted by: mamuller (IP Hidden, Junior member, 10)
Date: January 31, 2005 04:50AM
To recover active and healthy protein you need to use
a light staining solution, commassie is a protein hammer, which limit your protein recovery. use SB010 staining solution 10ng sensitivity, staining during process & and once you have location of protein can removed with 1 wash distilled water. The kindest protein healthy efficent recovery system is electro elution with GeBAflex-tube. See www.geba.org Protein from gel extraction Maurice
Re: eluding proteins fom PAGE gel
Posted by: bassamfahmawi (IP Hidden, Regular member, 42)
Date: March 15, 2005 03:50AM
There are sevaral methods for extracting proteins from electrophretic gels. Since you need to recover your protein in an active manner, the best stain for this purpose is the a negative stain (Zinc staining). You can check Bio-Rad, Pierce, and Sci-PLAS all these companies has elution appratus and methods. One thing worth mentioning here is that you can cut the band after negative staining and using any of the electroelutions cells provided by the above mentioned comapnies, or flash freeze this gel piece by liquid nitrogen and then crush genrly in suspend in PBS dialysis buffer to get the protin in most active possible amnner.
Good Luck!
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