Analogous to Dr. Mirella, we've used the ratio of A320/A280 (for membrane proteins). In response to a fussy reviewer, I located some relevant references (refs. 10-13, copied below) when we referred to this in "A high-throughput differential filtration assay to screen and select detergents for membrane proteins," Vergis et al., Anal. Biochem 407:1 (2010).
[10] S.J. Leach, H.A. Scheraga, Effect of light scattering on ultraviolet difference spectra, J. Am. Chem. Soc. 82 (1960) 47904792. [11] Y. Cordeiro, F. Machado, L. Juliano, M.A. Juliano, R.R. Brentani, D. Foguel, J.L. Silva, DNA converts cellular prion protein into the b-sheet conformation and inhibits prion peptide aggregation, J. Biol. Chem. 276 (2001) 4940049409. [12] G.J. Lee, A.M. Roseman, H.R. Saibil, E. Vierling, A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a folding-competent state, EMBO J. 16 (1997) 659671. [13] Y. Panyukov, I. Yudin, V. Drachev, E. Dobrov, B. Kurganov, The study of amorphous aggregation of tobacco mosaic virus coat protein by dynamic light scattering, Biophys. Chem. 127 (2007) 918. Regards, -MW Michael C. Wiener, Ph.D. Professor Department of Molecular Physiology and Biological Physics University of Virginia PO Box 800886 Charlottesville, VA 22908-0886 434-243-2731 434-982-1616 (FAX) On Fri, 21 Feb 2014 15:05:46 +0000 "Tanner, John J." <[email protected]> wrote: >I am aware of an assay for aggregation (aggregation rate) that is based on >fluorescence measurements. It involves excitation at 280 and emission in the >range 260-400. Is there a reference for the absorbance method? > >See > >Nominé Y, Ristriani T, Laurent C, Lefèvre JF, Weiss E, Travé G. A strategy for >optimizing the monodispersity of fusion proteins: application to purification >of recombinant HPV E6 oncoprotein. Protein Eng. 2001 Apr;14(4):297-305. PubMed >PMID: 11391022. > >http://www.ncbi.nlm.nih.gov/pubmed/11391022 > >Jack Tanner > >Sent from Jack's iPad > >On Feb 21, 2014, at 7:23 AM, "Vivoli, Mirella" ><[email protected]<mailto:[email protected]>> wrote: > >Dear Prerana, > >before starting the crystallization you could try to check the state of you >protein, simply determining the Aggregation Index (AI) from measuring the >absorbance at 280 nm and 340 nm. The AI is then computed using this simple >formula: AI= 100 x (Abs340/(Abs 280 - Abs 340)). Soluble and non-aggregated >proteins solutions typically have an Aggregation Index of 2 and lower, whereas >for some aggregation will be 2-5; heavily aggregated proteins show an >aggregation index >5. Of course you cannot use Nanodrop for it (because the >wavelength for the baseline normalization is 340 nm). I would try to decrease >the concentration of your protein to 4-5 mg/ml.Good luck. > >Best, > >Mirella > > >Vivoli Mirella > >Postdoctoral Fellow >University of Exeter, >Biosciences >Biocatalysis Centre, Henry Wellcome Building >Stocker Road, >Exeter, >Ex4 4QD >Tel:+ 44 (0)1392 726121 > > >________________________________ >From: CCP4 bulletin board >[[email protected]<mailto:[email protected]>] on behalf of Prerana G. >[[email protected]<mailto:[email protected]>] >Sent: Friday, February 21, 2014 11:14 AM >To: [email protected]<mailto:[email protected]> >Subject: [ccp4bb] > >Hi, Sorry for asking an off-topic question, >I have recently purified a protein having a molecular weight of 40kDa and >concentration of the protein was 8mg/ml. When I tried to set the protein for >crystallisation using micobatch method, the protein started precipitating in >most of the buffer conditions of Crystal screen and Peg ion. The precipitation >took place very quickly (within 5-10 mins). >How should I overcome this problem? > > >Regards >Prerana
