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" <m.viv...@exeter.ac.uk<mailto:m.viv...@exeter.ac.uk>> 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 [CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>] on behalf of Prerana G. [tracy...@gmail.com<mailto:tracy...@gmail.com>] Sent: Friday, February 21, 2014 11:14 AM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> 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
