Good evening all of you its nice to write hear my lab experience i.e. now become habbit to write this to me and now my aim is to make perfect my practical skill because to hone perfect my overall grade points. Now i stop hear blowing my own trumpet and start for topic
Thought there are several protein assays available,we do DNS last time but the most preferred one in many laboratories is "Lowry assay". It is effective in the concentration range of 0.1 mg/ml to 1 mg/ml.(we use 1 mg per ml i.e. add 50 u in 50 ml water )
As per procedure provide 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) (i goohled this )
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, simple and easy .
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. take 50 ml in 50 u BSA stock and
Then make sample o.1 mg per 1 ml of distilled water.
Then took 7 test tube mark them
B, 1,2,3,4,5, uk
Similarly, we can prepare various standard concentrations like 0,0.2,0.4,0.6,0.8,and add salivary amylase in unknown 1ml 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, 0.5 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 firest year.
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,
And after this we go to show results of optical density in calorimeter to sir ,sir amaze to see results( i thought results may
Incorrect because there may mistake in stock a d working solution we took 1mg per 1ml sample ) while thinking this sir say why this division results are correct we dont have answer if this but i get answer of my doubt that we can use BSA solution @ 0.1 mg per 1ml to 1 mg per ml both results may correct..
Hope, this helped you :) Found any mistakes? Doubts? Let me know with your messages
