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protein quantification

protein quantification - SDS-PAGE Gel Electrophoresis Forum

protein quantification - SDS-PAGE Forum and Gel Electrophoresis Forum. Discuss the running of agarose gels, sds-page gels, and other gels including sequencing, gradient or tricine.


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Old 09-04-2010, 01:31 AM
Pipette Filler
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Default protein quantification



Hi,
can any one let me know how to quantify proteins secretes by a fungus extracellularly/
thank you.........
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Old 09-07-2010, 03:41 PM
Pipette Filler
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Default Re: protein quantification

Quick and Dirty: UV 280 nm Absorption:
- dependend on protein mixture (there are only a few AS wich absorb
here) there is an "normal" extinction range between 0,5 and 4 for 1g/l protein. Is
the mixture compsosed of many different Proteins you can suggest an extinction of 1
for 1g/l protein.

ELISA: good and sensitive for 1 known protein you want to quantify. Protein can be in a
mixture and does not need to be isolated (but you must find special antibodys for
it). Alternatives a methods of biolabelling, but I think they are more useful for
protein mixtures.

Densiometric analysis:
If you have a goog gel, a good standard and a good analysing software you can
quantify proteins direct from gel (but you need minimum amount for detection).
Only useful with relative high amounts of proteins.

Colorimetric assay (Bradford, Biuret, etc):
One of the cheapest and standard method for protein quantifying is bradford
assay with coomassie briiliant blue 250. It changes emmission wavelength in
contact to 2 or 3 AS. Because of this it is not a high exact method (dependend
on handling 5-20 %! difference). It can be used for protein mixtures or single
proteins. Meanwhile there are so many different assays of this type available, so
check some companys about their products.

Affinity Chromatography:
Based on a standard you can get your protein amount from the resulting peak
area. Is to be used for single Proteins. It is a combination of purifiying and
Quantification.

Gel Chromatography:
Same as above, but gel based. Can be used for protein mixtures.

MALDI-TOF:
Like LC-MS/MS detection, but better for proteins. Can be used to detect a large
number of protein quantitys, where the single quantity of each protein is even of
interest.

NIRS, ICP-MS: Alternative MS methods.

ICAT:
Isotope coded affinity tags method. Is for quantitative proteomics. Won't get to
detail, just for information.

Okay in general, the selected method depends on your application. Are you going to determine quantities of a single protein or of a mixture? If it's a mixture, how complex is it (how many different proteins containing)? How great is the amount of mixture/solution you can analyse? How exact have to be my result ? Based on these questions you should select a method. Commonly you'll use any of the colorimetric assays, the UV, or densiometric method. Chromatography isn't so common just only for the purpose of quantifying. The MS Methods need a very expensive equipment and are mor used for high throughput or proteomics applications. Lastly I like to say that there might be a quantifying based on magnetic beads (when youre "fishing" proteins), but I'm not sure.
Hope I could help a little bit...
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