Assay Lowry

Cu ions from the Biuret reaction (see Section 1.2.1) form an unstable blue complex with the Folin-Ciocalteau reagent. This complex serves as a measure of the protein concentration. 

Disadvantages The Lowry assay requires a lot of pipetting work and many buffer components can interfere, including mercaptoethanole, HEPES, TRITON-X-100, and other detergents (they precipitate). Hence, it is advisable to precipitate the protein from the samples (see Section 1.5) and to perform the Lowry assay on the protein pellet. You have to make the reagents yourself, except for the Folin-Ciocalteau reagent available from (Merck). The Lowry protein values of different labs are difficult to compare, because every lab carries out the assay a little differently. 

Larson, E., et al. (1986). Artificial Reductant Enhancement of the Lowry Method for Protein Determination, Anal. Biochem. 155 243-248. 

Markwell, M., et al. (1978). A Modification of the Lowry Procedure to Simplify Protein Determination in Membrane and Lipoprotein Samples, , 4na/. Biochem. S7 206-210. 

---

Alternate Protocol 2 Microtiter Plate Modified Lowry Assay B1.1.6... 

The modified Lowry assay can also be done in a 96-well microtiter plate format. The assay has a working range of 1 to 1500 pg/ml. 

Protein determination by the Kjeldahl method is slow and very few samples can be run at one time. The Lowry and many other protein tests are much more convenient. A Lowry assay on a test tube rack of 40 samples can be done in <2 hr. 

Yeang, H.Y., Yusof, F. and Abdullah, L. (1998) Protein purification for the Lowry assay acid precipitation of proteins in the presence of sodium dodecyl sulfate and other biological detergents. Analytical Biochemistry 265, 381-384. 

Folin-Ciocalteu (Lowry) Assay. The quantitative Folin-Ciocalteu assay (also often called the Lowry assay ) can be applied to dried material as well as to solutions. In addition, the method is sensitive samples containing as little as 5 /Ug of protein can be analyzed readily. The color formed by the Folin-Ciocalteu reagent is thought to be caused by the reaction of protein with the alkaline copper in the reagent (as in the biuret test) and the reduction... 

For the preparation of carbonyl-free solvents see Section 5.3.2.2.) The cells are washed three times in serum-free medium and harvested by scraping. The harvested cells are washed again with PBS, pH 7.4. Cell disruption is effected by light sonication and after centrifugation (600 x g, 15 min) the insoluble material is removed. The supernatant fraction is retained and the protein concentration assessed by the Lowry assay. 

The Bradford assay is particularly suited to samples where there is a high lipid content which may interfere with the Lowry assay. 

With reagents prepared in advance, the Lowry assay requires 1 h. A 400-pL sample is required, containing 2-100-pg protein (5-250 pg/mL). Nonlinear calibration curves are obtained, due to decomposition of the Folin reagent at alkaline pH following addition to the sample that results in incomplete reaction. Interferences include agents that acidify the solution, chelate copper, or cause reduction of cop-per(II). 

Total protein in an unknown sample was estimated using the Lowry and Bradford assays. Results were 33 2 pg/mL from the Lowry assay and 21 1 pg/mL from the Bradford assay, using stock solutions of BSA as standards in each assay. The unknown sample was then thoroughly oxygenated, and the assays were repeated. The Lowry method yielded 22 1 pg/mL, while the Bradford results did not change. Why did the results of the Lowry assay decrease by 33% ... 

It is likely that interferences (Cu2+ reducing agents, possibly reducing sugars) are present in the first Lowry assay thorough oxygenation of the solution oxidizes these species, so that they do not interfere in the second assay. 

The Lowry assay [16] uses the reaction of cupric sulfate at alkaline pH in the presence of tartrate, producing a blue chromogen formed from four peptide bonds and one atom of copper. Addition of folin phenol reagent further enhances the color, with a maximum absorbance at 750 nm. The Lowry assay demonstrates the greatest sensitivity of the common protein concentration determination methods and varies only slightly when using the two common calibrators, BS A and BGG. Not surprisingly, this remains a very commonly used method. 

The final resuspension should be in a volume calculated to give an appropriate protein concentration approx 100 mg of membrane protein is obtained for every gram of brain tissue prepared. After the final resuspension, a sample of the homogenate is taken for determination of protein content by Bradford or Lowry assays. 

Mix test tubes thoroughly, then transfer a fraction of each sample to another set of test tubes to be assayed for protein content by the Bradford or Lowry assays. The fraction of the total sample must be adequate to obtain good results with the protein assay. 

Merino wool top was cleaned by sohxlet extraction using dichloromethane to remove fatty matters and repeated washing with cold water. Hie wool was air dried at about 40°C. The wool proteins were extracted overnight from wool by an extensive treatment with 8M Tris/urea buffer containing (SO mM) dithiothreitol as reduction chemical at pH 9.3 and 25 °C. The reaction was stopped using 20% iodoacetamide. The wool proteins were dialysed against distilled water for 4 days. The protein content of the enzymes and the wool hydrolysate were determined using the Lowry assay [6]. 

Protein, iron catd labile sufiir analysis Protein was quantified by a modified Lowry assay (12) on triplicate samples at 3 different sample amounts. Iron was analyz by atomic absoiption using deuterium arc background correction by the method of standard additions. AU samples for iron analysis were passed over Chelex resin io>Rad) to remove any unbound iron. Labile sulfur analysis was done according to the method of (13) except that CHQ3 was used for extraction of pigment acidification products. 

The subunit structure of the RC is still unceitain. The inequalities from protein to protein in the Lowry assay as well as the uncertainty of the P800 differential extinction coefficient make an... 

A. Lowry assay (Lowry et al., 1951) Folin-Ciocalteu phenol reagent (Merck). 

Pro The Lowry assay exhibits the best accuracy with regard to absolute protein concentrations due to the chemical reaction with polypeptides. This contrasts with the other two methods, which, as dye binding assays, exhibit more variation depending on the different reactivity of the given proteins (standards as well as samples). 

In the case of photometers/fluorometers operating with filters (usually microplate readers) the correct wavelength may not be available. Instead, a similar wavelength may be employed Lowry assay 500-800 nm, Bradford assay 540-620 nm, dot-blot assay 380-450 nm (excitation), 450-520 nm (emission). In the case of fluorometry allow for a sufficient wavelength interval between excitation and emission (consult the operating instructions of your instrument). 

---