he affinity chromatographic purified protein were subjected to DLS-Size distribution and the Z average values was 189.9,643.9,669.7,625.1 and 811.8, from this results how to find the molecular weight ?
If the numbers you quoted are in nanometers, then you are either working with an aggregated protein (or at least a solution of protein that is partially aggregated), or a fibrous protein (collagen, for example). Typical globular protein sizes are less than 10 nm in diameter.
Some DLS software can be used to calculate molecular weight based on the size measurement, but this probably won't be very accurate for a heterogeneous, aggregated sample.
If you have access to a SEC-MALS system, you can get a more thorough understanding of the size distribution and particle masses.
thanks sir,
how the SEC-MALS is differes from DLS ,it is new to me
SEC = size exclusion chromatography, separation on HPLC based on molecular size and shape, combined with
MALS = multiangle light scattering. Uses in-line refractive index and multiangle light scattering detectors. Determines molecular mass as particles pass through, requiring no calibration standards.
http://www.wyatt.com/solutions/techniques/sec-mals-molar-mass-size-multi-angle-light-scattering.html
Do you mind me asking what DLS instrument you are using?
The Malvern Zetasizer can perform a Mw measurement using static light scattering by measuring the intensity of scattered light for a number of different concentrations of your sample.
The same measurement will also inform you of the A2 parameter which can be used to quantify the stability of the sample.
Thanks Mr.Alexander, i tested my samples with Zetasizer Ver 7.11 , whether this instrument can measure?
please provide the protocol >
A typical chromatography setup should typically not pass any particles larger than 100nm in size, so it is a little surprising to read your report of several hundred nm z-average size. Typical proteins are smaller than about 10nm. This would imply that your samples are aggregated in the preparation you measured. You could try centrifugation or filtration [20nm Anotop filters might work] to check if the z-average reduces significantly.
It is possible that your protein concentration is too low for detection.
Molecular weight measurements with the Zetasizer may be performed by preparing different known concentrations of your sample (using the molecular weight SOP) however your samples would have to be more homogenous to provide meaningful answers with that method. Details about how to do it can be found in the Manual, available at Start - Programs - Malvern Instruments - Zetasizer software - Manuals - Man0485-1.1 (Zetasizer Nano user manual).pdf in the Chapters 5, 8, and 12 ( online at http://www.malvern.com/en/support/resource-center/user-manuals/MAN0485EN.aspx )
http://www.materials-talks.com/blog/2016/01/21/zetasizer-sensitivity-for-protein-dls/
http://www.materials-talks.com/blog/2014/09/16/faq-molecular-weight-estimate-from-dls/
Can I study a sample with more than one molecular using the Zetasizer?
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