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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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Purification of enzymatically active protein from PAGE
Posted by: monsa (IP Hidden, New member, 9)
Date: October 4, 2005 11:59AM
I am trying to purify the digested product of His-tagged fusion proteins (pET System – Novagen) produced in E. coli (BL219DE3)) to compare their kinetics of different isoforms of an oxidative decarboxylase (soluble, malic enzyme, E.C. 1.1.1.40). My original idea was to His purified the fusion proteins, digest them, run them again through the affinity column and get rid of the enterokinase.
The problem I first encountered was that not only the full length (100 kD) but also truncated versions, of much lower MW (between 20-50 kD) are produced. I tried to fix this problem using host strains that carry rare codon tRNAs (Rosetta2 – Novagen) but without success. I was thinking that a good way to obtain the digested product and get rid of the rest (i.e., uncut fusion, truncated forms, enterokinase) would be to run an exclusion chromatography after digestion to separate the species by size. This would most likely work for the truncated forms and enterokinase since they are much smaller than the fusions, at a cost of protein loss. On the other hand, somebody suggested to run a preparative SDS-PAGE and then elute the cut the right “band” and elute the protein. Would it be possible to get good quality protein using this procedure and have “publishable” results? I wonder if this would be an acceptable procedure to obtain and analyze protein kinetics. What protocol for protein elution from SDS-PAGE (or native?) and subsequent refolding would you reccommend?
Re: Purification of enzymatically active protein from PAGE
Posted by: shane (IP Hidden, Junior member, 10)
Date: October 9, 2005 08:04AM
Hello Monsa,
Electro elution of protein of gel is a very well accepted procedure and u can use this for ur work without any hesitation, infact many companies provide this electroelution device. or u can arrange urs own inhouse. if ur so worried about sds u run native and get native protein in appropriate buffre. bye thank u
Re: Purification of enzymatically active protein from PAGE
Posted by: shane (IP Hidden, Junior member, 10)
Date: October 9, 2005 08:04AM
Hello Monsa,
Electro elution of protein from gel is a very well accepted procedure and u can use this for ur work without any hesitation, infact many companies provide this electroelution device. or u can arrange urs own inhouse. if ur so worried about sds u run native and get native protein in appropriate buffre. bye thank u
Re: Purification of enzymatically active protein from PAGE
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: October 10, 2005 08:05AM
shane, I would be very interested to know how you made your own electroelutor. Probably I can follow it if it is not too difficult. Sometime I put the gel piece in a buffer on ice and chop it into tiny pieces. I can elute certain portion of the protein out of the gel but I don't know exactly how much recovery I got. It's not recommended for quantitative analysis but is ok for non-quantitative activity detection.
Re: Purification of enzymatically active protein from PAGE
Posted by: mamuller (IP Hidden, Junior member, 10)
Date: November 29, 2005 08:21AM
Protein PAGE purification, Use GeBAflex-tube electro elution, insert the gel part containing the protein, add same buffer for electro elution inside the tube, electro elute for x minute, reverse polarity for 1-2 min, remove gel fragment, dialysis process for upto 60min, a very simple methode, review website at www.geba.org for details and method, minimum garantied is 70% protein recovery, the same GeBAflex-tube can then be used for dialysis removing SDS. At www.biowww.net search gebaflex-tube and you find customer appreciation. feel free to e-mail, if more info is needed, Best regards Maurice
Re: Purification of enzymatically active protein from PAGE
Posted by: sreenathkonanki (IP Hidden, New member, 2)
Date: September 29, 2007 01:21AM
hi
this is sreenath from india i want information on purification of enterokinase and at the same time charecterization of enterokinase please help me thanks & regards sreenath konanki
Re: Purification of enzymatically active protein from PAGE
Posted by: sreenathkonanki (IP Hidden, New member, 2)
Date: September 30, 2007 09:31PM
Hi
this is sreenath konanki i need information on protein purification i want to do PhD if any body can please help me in this regard thanks & regards Sreenath Konanki sreenathkonanki@gmail.com Mobile: +919849515868 Hyderabad India
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