Académique Documents
Professionnel Documents
Culture Documents
Swetha
Lowry protein assay is a biochemical assay for determining the total level of proteins in a solutions. This method is named on the name of biochemist OLIVER .H.LOWRY .
Wu first proposed the use of folin phenol reagent in 1922. In 1940, O.H.LOWRY devised and modified the folin reagent.
In this reaction protein binds to copper (Cu2+) in alkaline medium and produces Cu1+-protein complex. In the second reaction complex catalyses oxidation of aromatic amino acids by reducing Phosphomolybdotungstate to heteropolymolybdenum blue. This reaction produces strong blue colour,which predominantly depends upon tyrosine and tryptophan content of protein .
s.no 1.
Reagents Alkaline copper reagent 2% Sodium carbonate in 0.1 N NaOH (washing soda or soda ash) 2 % Na-K tartarate (Rochelle salt)
Function
This ingredient maintains cupric ions in solution at an alkaline pH by stabilizing the chelate complex. This provides the Cu (II) ions which form the tetradendate chelate complex. Cu (II) ions give the reagent its characteristic blue color. In the absence of copper, color intensity would be determined primarily by the tyrosine and tryptophan content of the protein, and to a lesser extent by cysteine and histidine. Copper(II) enhances color formation by chelation with the peptide backbone, thus facilitating the transfer of electrons to the chromogens.
Cu2SO4 (1%)
Reagents
2.
Function
Phosphomolybdic acid
Conjugated, unsaturated compounds ( in this case, copper chelated protein complex) reduce PMA to molybdenum blue. PTA is less sensitive to reduction as compared to PM.
Phosphotungstic acid
Phosphomolybdic & phosphotungstic components in FC reagents gets reduced by certain amino acids present in the protein like tyrosine , tryptophan thereby to their respective oxides. Estimation of this compound in turn provides the amount of protein present in the sample.
PREPARATION OF REAGENTS
1. PREPARATION OF STOCK BSA (1mg/ml, 10ml) 10mg of BSA was weighed and taken in a falcon. Then, 10ml of distilled water was added to it. 2. PREPARATION OF WORKING BSA (100g/ml, 10ml) From stock BSA 1ml was taken and 9ml of distalled water was added to it. 3. PREPARATION OF ANALYTICAL REAGENT 1% Copper Sulphate 0.1 gm of CuSO was weighed and taken in a flask. Then, distilled water was added to it to make up the final volume to 10ml. 2% Sodium-Potassium Tartarate - 0.2gm of Na-K Tartarate was weighed and taken in flask. Then, distilled water was added to it to make up the final volume to 10ml.
Normality
0.1 N NaOH = wt. of solute x 1000 40 x 300 wt. of solute = 0.1 x 12 = 1.2 g 6g of NaCO was weighed and taken in flask. Then, final volume was made to 300ml by 0.1 N NaOH.
Then, analytical reagent was prepared by adding Na-K Tartarate : CuSO : Alkaline Reagent in the ratio of 0.5 : 0.5 : 49 For 300ml of Analytical reagent 1.5ml Na-K Tartarate, 1.5ml CuSO and 297ml of alkaline reagent was added. These are to be added in a specific order only. Na-K Tartarate is the first to be added followed by CuSO, then alkaline reagent.
RESULT
STANDARD CURVE OF BSA BY LOWRYS METHOD
0.6
0.5
Absorbance at 660nm
0.4
y = 0.009x
0.3
0.2
0.1
10
20
30
40
50
60
70
From Graph, y = 0.009x where, y = absorbance of the sample x = concentration of protein in the sample For unknown 1 ( 0.5ml sample ) y = 0.096 Therefore, x = 0.096 / 0.009 x = 10.66 g/ml
Therefore, in 1ml sample, concentration of protein = 10.66 x 2 = 21.32 For unknown 2 ( 1ml sample ) y = 0.181 Therefore, x = 0.181 / 0.009 x = 20.11 g/ml
PERCENT ERROR
Percent Error = Actual Conc. Obtained Conc. X 100 actual conc. = 20 20.71 x 100 20 = 0.71x100 20 = 3.55
1 to 10 mg/ml Several folds less 100 times less sensitive than Lowry's sensitive than Lowry's
Protein Digestion
BIURET TEST No NINHYDRIN TEST Yes LOWRYs No
Have to be linearized
Adaptability : Lowrys assay is adaptable to both test tubes and microtitter plates Less protein to protein variation than dye binding Bradford assay.
PRECAUTIONS
Extra heat is liberated in the first few minutes after FR addition which may lead to dissociation of Phospho Molybdate. Hence the reaction has to be buffered well. Mixing of the assay mixture after FR addition: The drastic difference in the ph of Analytical reagent and FR may lead to dissociation of phosphomolybdic acid which may cause uneven colour formation. The assay reaction is optimum at pH 10. So buffering is essential.