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Protein quantification using HPLC/fluorescence

HPLC fluorescence

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3 replies to this topic

#1 phub11

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Posted 10 May 2016 - 07:06 PM

Hi,

 

I am wondering if anyone on here has experience using native fluorescence/HPLC to quantify protein concentration. Up until now we've been using ELISAs as the protein quantities are in the ng/pg range, but obviously HPLC would make things much easier/quicker. The proteins we are assaying are about 30 kDa., and have the expected number of aromatic residues.

 

We have an Aglient 1100, and I'm looking at purchasing a used fluorimeter to eliminate the need for doing ELISAs; however, given that we can't label the proteins (I'm assuming variations in labeling efficiency between the calibration standards and the assayed samples would be too great) I'm concerned whether native fluorescence would be sufficiently sensitive. Is this likely to be the case?

 

On a related note, is there a web server that can analyze amino acid sequence and predict improvement in signal when comparing native fluorescence to absorbance at 220nm?

 

Any feedback would be greatly appreciated.



#2 mdfenko

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Posted 12 May 2016 - 04:39 AM

are you looking at a purified protein, a mixture or a specific protein within a mixture?

 

if you're looking to quantify a purified protein or whole mixture then you may want to look into IR detection like the emd-millipore direct detect IR spectrometer.

 

if you're trying to quantify a specific protein in a mixture then you may try lc/ms.

 

if you insist on trying direct fluorescence then you may have to determine the intrinsic fluorescence and proper excitation and emission wavelengths of the purified protein with a scanning spectrophotofluorimeter.


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#3 phub11

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Posted 14 May 2016 - 06:07 PM

Many thanks for your reply. Yes, I'm trying to quantitate a specific protein from a mixture. I've found a paper that might be helpful to future viewers of this posting:

 

http://www.ncbi.nlm....pubmed/22809792

 

It's not a perfect answer to my question, but suggests that the LOD is in the low nanograms for the proteins I am interested in (under the authors' conditions).

 

The Agilent 1100 fluorescence detector appears to offer tools to help select optimal ex/em wavelengths - which is a nice feature.

 

Thanks again!



#4 Bennie George

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Posted 24 October 2017 - 10:08 PM

How about Nuclear Magnetic Resonance Spectroscopy?







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