Hi,

Two things i would like to add:

1) Due to the dependence of A280 on amino acid composition, a simple 
two-wavelength 280 and 340 or 320 comparison is not very ideal for 
determining the scatter component of the UV absorption of protein/protein 
aggregate particles (the usefulness of this simple method for determining 
the degree of aggregation is not questioned). Instead, we use data points in 
the 320-350nm range from a UV absorption/scattering scan to plot a curve for 
determining the scattering component, which serves two purposes: a) to be 
used for subtracting the A280 to get real UV absorption of the protein at 
280nm, b) to have an idea of how aggregated the sample is.

To do this, we plot a double log curve with absorption values A and 
wavelengths lambda at 320 330 340 350 nm, and fit it to the equation
log(A)=C - k*log(lambda)
Then the resulting equation can be used for extrapolating the scatter 
component at 280 nm. Of course the larger this component is, the more 
aggregated the protein is. The degree of aggregation (particle size and 
population) is also reflected by the k value. The larger the k is, the less 
aggregated the solution is.

An explanation of the theory behind this can be found here:
http://www.chem.agilent.com/Library/applications/59633927.pdf
Our favorite method is the one in the "scattering model" part, note that in 
figure 7 their molecule has UV absorption peak at 300, while in most of our 
cases, the proteins have peak at 280. There should be an academic article on 
this method. However I can not locate it right now. Apologies for that.

In fact, a quick look at the UV curve in the 300-350 nm range can directly 
tell us how badly aggregated a protein solution is. A perfectly not 
aggregating protein solution should have a UV absorption curve that quickly 
drops to nearly zero at 300 nm. One can test this by measuring an 
aggregation sample before and after filtration or centrifugation and see the 
dramatic change on the UV curve.  (0.22um filter's pore sizes are quite 
comparable to the 200-400nm UV wavelengths, while most globular proteins are 
roughly 2-10 nm in diameter.)


2) Nanodrop's baseline function in the scan mode ("Protein 280" for example) 
is actually an all-point zeroing. At the blanking step, the instrument 
records a blank absorption curve and later uses this curve to subtract the 
sample curve. So when using a proper solution as blank, the sample curve is 
quite faithfully reflecting the UV absorption + scattering. I believe most 
UV spectrometers allow the users to do the same in the scan mode. So they 
are all good for this task.

Zhijie



-----Original Message----- 
From: Michael C. Wiener
Sent: Friday, February 21, 2014 12:16 PM
To: [log in to unmask]
Subject: Re: [ccp4bb] Aggregation assay

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) 4790–4792.
[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) 49400–49409.
[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) 659–671.
[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) 9–18.

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." <[log in to unmask]> 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" 
><[log in to unmask]<mailto:[log in to unmask]>> 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 
>[[log in to unmask]<mailto:[log in to unmask]>] on behalf of Prerana 
>G. [[log in to unmask]<mailto:[log in to unmask]>]
>Sent: Friday, February 21, 2014 11:14 AM
>To: [log in to unmask]<mailto:[log in to unmask]>
>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