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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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ScFv-H6 precipitation after dialysis
Posted by: Alesia (IP Hidden, Unregistered user, )
Date: March 24, 2005 12:29PM
dear all,
I purified ScFv (pI=7.6) on the His-Tag column. My protein was eluted with 100mM imidazole. After I saw the thick pure bands on my SDS PAGE. Also I measured the 280 nm and noted the good protein amount. After I dialysed o/n in dialisys buffer contained 50mM Tris pH 8.0, 50 mM NaCl, and almost all had precipitated. From what parametres you can advise me to begin the test the conditions for avoid this precipitation? I should note, that for my ElISA assay i can use even the protein non-dialysed, but for gel-exclusion chromatography I should obtain conditions to have a soluble protein, for it will not precipitate in the column once separated from imidazole. Thank you very much for answers
Re: ScFv-H6 precipitation after dialysis
Posted by: bassamfahmawi (IP Hidden, Regular member, 42)
Date: March 24, 2005 02:33PM
Hello,
This a very common problem with expressed fusion proteins. However, to overcome this problem during dialysis you need to perform several chnages of your dialysis buffer and then dialize over noght. I have found this procedure quit successful in obtaining my His-tag protein without precipitation. You need to change your dialysis buffer every 30 minutes (3 times and 2 times for one hour each after the last change you can proceed with your dialysis for overnight. Good Luck!
Re: ScFv-H6 precipitation after dialysis
Posted by: Irina (IP Hidden, Unregistered user, )
Date: March 28, 2005 03:21PM
Hello AlesiaĦĦĦĦ:
I´ve got exactly the same problem. I´ve tried with glycerol 7% in dialysis buffer but I haven´t got any result. Did you try bassamfahmawi method? Thank you for your answer Irina
Re: ScFv-H6 precipitation after dialysis
Posted by: David G. (IP Hidden, Unregistered user, )
Date: April 15, 2005 03:53AM
Look at this article :
Hybrid Hybridomics. 2003 Dec;22(6):347-55. Related Articles, Links Effect of imidazole on the solubility of a his-tagged antibody fragment. Hamilton S, Odili J, Pacifico MD, Wilson GD, Kupsch JM. RAFT Institute of Plastic Surgery and Cancer Research Trust, Gray Cancer Institute, Mount Vernon Hospital, Northwood, Middlesex HA6 2RN, UK. A number of studies have demonstrated the limited solubility of single-chain Fv antibody fragments and its improvement by genetic engineering. This limits the stability of recombinant protein upon storage and the efficiency of chemical modification. The RAFT3 scFv used in the present work is specific for melanoma-associated proteoglycan and an attractive candidate for clinical radioimaging studies because of its unusual radiolabelling properties. However, when expressed with a c-terminal his(6) IMAC purification tag, the recombinant protein starts to precipitate after column elution and dialysis against PBS and reaches a concentration of soluble protein of approximately 150 microg/mL within a few days upon storage at 4 degrees C. We tested several commonly used buffer modifications (addition of detergents, high salt, amino acids) to improve the solubility and stability of the protein but without any major improvement. However, we found that, when the final dialysis step was omitted and the protein left in IMAC column elution buffer (PBS containing imidazole), it remained soluble. Furthermore, several months old and precipitated protein could be redissolved in this buffer without loss of antigen binding. This observation and the largely pH-independent nature of protein solubility suggest that neither salt bridges formed by the his(6) tail nor cross-linking of his(6) tails mediated by metal ions leached from the column during elution are responsible for the limited solubility of the protein in the absence of imidazole. The presence of imidazole did not interfere with radiolabelling and in vivo tumor targeting in a mouse model. The solubilizing and stabilizing effect of imidazole could be of use for his(6) tagged and poorly soluble recombinant proteins other than scFvs. PMID: 14683594 [PubMed - indexed for MEDLINE]
problem of host proteins
Posted by: rani (IP Hidden, Unregistered user, )
Date: April 19, 2005 06:10AM
i want to purify an enzyme transglutaminase 47 kd using IMAC.i first used GJ1158 as the host and the expression was very good but on purification by IMAC the host proteins are also seen in SDS-PAGE it seems to be eluting along with my protein .help please
Re: problem of host proteins
Posted by: bassamfahmawi (IP Hidden, Regular member, 42)
Date: April 25, 2005 01:15PM
Hello,
Are these proteins co-purifying consistantly with your protein? Anyways, Since you are using IMAC, I am assuming that your protein is His tagged, if yes at what Imidazole conctration your protein elute? From my personal experience washing several times with 20 up to 40 mM imidazole will reduce the non-specific binding of other proteins and your protein can purify with significan purity when eluted with 100-25 mM imidazole. Also, if you are using Ni-NTA resin they are known of theri relative non-specific binding characterstics, therfore new resins have emerged which to my experince I have found the NI-TED resins to be much better resolving this problem and they are available through Machery-Nagel and Active Motif. Good Luck!
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