haiduc - 16-10-2006 at 14:23
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
chemoleo - 16-10-2006 at 14:55
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
solo - 16-10-2006 at 16:02
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 7848 times
haiduc - 16-10-2006 at 23:31
Aye, it was diluted 100 times, so those numbers stand for:
10%
30%
50%
70%.