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: CCP4BB@JISCMAIL.AC.UK
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." <tanne...@missouri.edu> 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"
<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
