haiduc
Harmless
Posts: 3
Registered: 5-4-2006
Location: Cracovia Poland EU
Member Is Offline
Mood: cloud nine 
|
|
Lowry protein assay: analysis
Hello!
I did the Lowry protein assay, I measured the absorbance at 500 nm, and what I got was:
water: 0,17
serum albumin 0,1%: 0,162
0,3%: 0,240
0,5%: 0,403
0,7%: 0,421
blood plasma: 0,590
albumin 100%: 0,563
All I need is a piece of advice on how to prepare a standard curve of absorbance vs. micrograms protein.
I use a linear regression constantly, though I have no idea how to relate all the data this time!
Thanks for your concern,
Haiduc
*_viens, viens chez moi, Voyageur
|
|
|
chemoleo
Biochemicus Energeticus
Posts: 3005
Registered: 23-7-2003
Location: England Germany
Member Is Offline
Mood: crystalline
|
|
Problem is, it isn't linear at all, right? at 0.7%, the AU is 0.421.
But at 100%, it is 0.563 !!!!  (correct me if I am wrong, but isn't 100% albumin a solid?)
Essentially, it means you have to dilute your samples such that it's absorbance is between 0.24 and 0.4. Before that, I'd advise to do your standard
curve again, but at a much greater resolution (more datapoints).
Also, which Lowry assay is it? There are many types.
Check there aren't any interfering compounds.
'While widely used, the Lowry procedure is less preferable an assay than some other protein assays since it is more subject to interference by a wide
variety of chemicals. Among the chemicals reported to interfere with the Lowry procedure are barbital, CAPS, cesium chloride, citrate, cysteine,
diethanolamine, dithiothreitol, EDTA, EGTA, HEPES, mercaptoethanol, Nonidet P-40, phenol, polyvinyl pyrrolidone, sodium deoxycholate, sodium
salicylate, thimerosol, Tricine, TRIS and Triton X-100.'
From http://www.animal.ufl.edu/hansen/protocols/LOWRY.htm
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
|
|
|
solo
International Hazard
Posts: 3975
Registered: 9-12-2002
Location: Estados Unidos de La Republica Mexicana
Member Is Offline
Mood: ....getting old and drowning in a sea of knowledge
|
|
Reference Information
A Simplification of the Protein Assay Method of Lowryetal. Which is More Generally Applicable
GARY L. PETERSON
ANALYTICAL BIOCHEMISTRY 83, 346-356 (1977)
Abstract
Some recent modifications of the protein assay by the method of Lowry, Rose- brough, Farr, and Randall (1951, .I. Biol. Chem. 193,
265-275) have been reexamined and altered to provide a consolidated method which is simple, rapid, objective, and more generally
applicable. A DOC-TCA protein precipitation technique provides for rapid quantitative recovery of soluble and membrane proteins from
interfering substances even in very dilute solutions (< 1 pg/ml of protein). SDS is added to alleviate possible nonionic and
cationic detergent and lipid interferences, and to provide mild conditions for rapid denaturation of membrane and proteolipid
proteins. A simple method based on a linear log-log protein standard curve is presented to permit rapid and totally objective
protein analysis using small programmable calculators. The new modification compared favorably with the original method of Lowry ef
al.
Attachment: A Simplification of the Protein Assay Method of Lowryetal. Which is More Generally Applicable.pdf (728kB)
This file has been downloaded 7983 times
It's better to die on your feet, than live on your knees....Emiliano Zapata.
|
|
|
haiduc
Harmless
Posts: 3
Registered: 5-4-2006
Location: Cracovia Poland EU
Member Is Offline
Mood: cloud nine
|
|
Aye, it was diluted 100 times, so those numbers stand for:
10%
30%
50%
70%.
*_viens, viens chez moi, Voyageur
|
|
|
|
![[*]](images/xpblue/default_icon.gif){kind=icon}
