Thursday, October 24, 2013

Lowry Assay Principle and procedure

Though there are several protein assays available, the most preferred one in many laboratories is "Lowry assay". It is effective in the concentration range of 0.01 mg/ml to 1 mg/ml. And, as an additional info, the paper published describing the procedure and principle of Lowry Assay is the most cited paper in the scientific history. (Feeling like, "Wow! I want to publish one to compete with Oliver.H. Lowry"???  :P)

Why Lowry? 

Though there are several other protein assays, mostly Lowry assay is used in many laboratories. The reasons for preferring Lowry are: sensitivity of the assay, highly reproducible, cost effective, easy to perform. Biuret assay is generally used for higher protein concentrations like tissue samples but, Lowry for less concentrated samples and hence used in most of the molecular biology laboratories where there will be need for assaying comparatively less concentrated protein samples (in most cases where we attempt to produce enzymes).
Other assays like Bicinchoninic Acid and Dye binding assays are used in only specific circumstances and mostly not preferred due to its sensitiveness to contaminants like carbohydrates, lipids, etc.,

 Principle behind Lowry's Assay for protein with procedure

The reactions that occur in Lowry assay are binding of Copper to the Nitrogen in the peptide. And, the phosphomolybdic tungstic acid in the Folin Ciocalteau reagent gets reduced to hetero poly-molybdenum blue by the copper catalyzed oxidation of aromatic amino acids in the peptide, in alkaline conditions. The assay must be done at the pH of 10 to 10.5 as it is sensitive to pH changes.


Reagents Required:

A) 2% of sodium Carbonate (50 ml) + 0.1 N NaOH solution (50 ml)
B) 10 ml of 1.56 CuSo4 + 10 ml of 2.37% Sodium potassium tartarate

Lowry's Reagent = 2 ml of (B) + 100 ml of (A)
Folin's Reagent what we use in our laboratory is ready made one (2 N) which is just diluted and made as 1 N for use. (by mixing with equal volume of water)

For constructing Standard curve, BSA is generally used. Stock of 1 mg/ml is required. 

Note: Prepare all the reagents in distilled water. And, prepare the reagents freshly before use, just before use, to avoid precipitation of the salt added, also mix the reagents A and B only before use. 

Procedure:
  • First the BSA stock is diluted for standard curve construction. Let the total volume be 1 ml, so for preparing a concentration of 0.05 ml, take 0.05 ml of 1 mg/ml BSA stock and mix with 0.95 ml of distilled water.
  • Similarly, we can prepare various standard concentrations like 0.1,0.2,0.4,0.6,0.8,1 mg/ml for  total volume of 1 ml by mixing 0.1, 0.2,0.4,0.6,0.8,1 ml of stock BSA with 0.9,0.8,0.6,0.4,0.2,0 ml of distilled water respectively. 
  • From this prepared standard concentrations of BSA, 0.2 ml must be taken for assay. For example, from the 1 ml of 0.05 mg/ml BSA prepared 0.2 ml must be taken in a separate test tube. Similarly, 0.2 ml must be taken from all the other standards.
  • Then, 2 ml of Lowry's reagent must be added to each of this 0.2 ml sample and incubated for 10 minutes.
  • Then, 0.2 ml of Folin's reagent is added to each of the incubated tubes and incubated for 30 minutes.
  • After 30 minutes, the tubes will have blue colored solution, which is further read at  660 nm in Spectrophotometer. 
  • Graph should be plotted with absorbance in y axis and concentration in X axis. 
And, using this standard graph, we can determine the concentration of unknown sample by extrapolation.

This is a very easy assay which I learnt in the very beginning  of my course, but, still a very useful one and I'll be using it even after years I believe. 

You know, I feel the major disadvantage of this Lowry assay is that you need to spend at least two hours to complete it including reagent preparation and incubation. (Sometimes, I feel like, oh, no, incubation for 30 minutes!How good it would be if there is no need for incubation?) And, when you don't have that much time, what will you do for assaying your protein? 

It's simple, just measure absorbance at 260 nm and also 280 nm. Using the formula,

Protein (mg/ml) = 1.55 *(Absorbance at 280 nm) - 0.76 * (Absorbance at 260 nm).

This can be used when you have a spec which operates in UV range. No need for doing Lowry Assay, Yippee :P 

Hope, this helped you :) Found any mistakes? Doubts? Let me know with your comments!

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