[RASMB] Help with Small Self-Associating Peptide

HGSR (Holger Martin Strauss) hgsr at novonordisk.com
Mon Dec 12 01:10:10 PST 2011


Hi Peter,

in addition to the comments by John, some more from my experience, mainly from SE:

- purity is absolutely essential, esp. when working with synthetic peptides. Ideally, measure the integrity of the peptide with MALDI-TOF before and after the experiment.

- if at all possible, measure the vbar, the true value of your peptide will likely deviate from the one Sednterp gives you. If you're only after the stoichiometry of the complex, it might be sufficient to stick with sigma (the lower limit of which you can define from the data) and ratios thereof.

- you can check the reversibility of your reaction from plots of lnC/r2 at different starting concentrations.

- the stoichiometry of the complex is more a problem of accuracy, so you need the best data you can get; scan many replicates, use long solution columns (200 microL and more), clean your lamp before the experiment, use thoroughly dialysed buffer (2800 g/mole can be tough, though). The decision wether your smallest sigma is really a monomer or a dimer is easier to answer than wether you have a hexa- or a heptamer.

- try to measure the baseline absorbance. If you do have a hexamer, you should be able to clear the meniscus with long solution columns at the highest speeds. You can get the baseline for all wavelengths used from this condition.

- if you can clear the meniscus, interference has clear advantages over absorbance (better S/N, mostly) and will help you to define the upper limit of the association. Be careful to measure a good water blank.

- use a buffer of sufficient ionic strength. Some peptides carry surprisingly high charge densities.

- if the peptide unfolds at low concentrations, degradation can be a real problem. If so, excluding as far as possible all sources of microbial contamination can help, including rinsing your cells with 70% ethanol.

Best, Holger

________________________________
From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On Behalf Of Peter Edward Prevelige Jr
Sent: Friday, December 09, 2011 11:57 PM
To: rasmb at rasmb.bbri.org
Subject: [RASMB] Help with Small Self-Associating Peptide

Hi All -

Can you give me some advice on the best way to analyze a small (~2800 Da) peptide that we think is self associating. The CD shows increasing helicity with concentration which continues to increase even at 10 mg/ml meaning we will likely have to record data at a less than ideal wavelength (240 nm) Our interest is really in determining the stoichiometry of the species formed at high concentration  (we suspect a hexamer), we care less about Kd. Looking through the archives I've seen conflicting suggestions, (ie. sed velocity vs sed equilibrium).  I was thinking about mimicking the approach in the Braswell/Holtzer paper but the peptide is somewhat valuable and limited so I wanted to ascertain whether or not this was the optimal way to proceed.

Thanks!

Peter


Peter E. Prevelige Jr.
Professor
Dept. of Microbiology, BBRB 416/6
Univ. of Alabama @ Birmingham
845 19th St. South
Birmingham AL. 35294-2170
Phone 205 975-5327
FAX 205 975-5479
prevelig at uab.edu<mailto:prevelig at uab.edu>
http://www.microbio.uab.edu/faculty/prevelige/prevelige-p.htm

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