7.

13 Protein Estimation by Lowry’s Method 113

22. This process if followed 4 times, the resultant faeces solution would become
homogenous. Transfer this mixture to 500 mL volumetric flask and make up the
volume.
23. Weigh the remaining feed in diet boxes and record in the notebook.
24. Determine N in urine and faeces by taking out 25 mL sample of urine and
50 mL sample of faces by micro-Kjeldahl method. Samples are analysed in
duplicate.
25. Calculate the total amount of N excreted in urine and in faeces by each rat
during balance period.
Determination of metabolic and endogenous nitrogen: Feed a separate group
with 4% freeze-dried, ether-extracted egg protein as the protein source. Since egg
protein at this level is completely utilized by rats, nitrogen in the urine and faeces
must be of endogenous origin.
Collect the determined nitrogen in the urine and faeces as described, and
incorporate in the calculations.

Calculations
N retained I  ðF  Fk Þ  ðU  Vk Þ
NPU ¼ ¼
N intake I
N absorbed I  ðF  Fk Þ
D¼ ¼
N intake I
and

N retained I  ðFk Þ  ðU  Uk Þ
BV ¼ ¼
N absorbed I  ðF  Fk Þ

where I is the intake nitrogen (nitrogen in the diet), F ¼ faecal nitrogen,

Fk ¼ endogenous faecal nitrogen, U ¼ urinary nitrogen and Uk ¼ endogenous
urinary nitrogen.

7.13 Protein Estimation by Lowry’s Method (Lowry et al. 1951)

Protein can be estimated by method described by Lowry and colleagues. The

method is sensitive enough to give a moderately constant value and hence largely
followed. Protein content of enzyme extracts is usually determined by this method.

Principle
The blue colour developed by the reduction of the phosphomolybdic-
phosphotungstic components in the Folin-Ciocalteau reagent by the amino acids
tyrosine and tryptophan present in the protein plus the colour developed by the
114 7 Qualitative and Quantitative Estimations of Amino Acids and Proteins

biuret reaction of the protein with the alkaline cupric tartrate are measured in the
Lowry’s method.

Reagents
1. Reagent A: 2% sodium carbonate in 0.1 N sodium hydroxide.
2. Reagent B: 0.5% copper sulphate (CuSO4
5H2O) in 1% potassium sodium
tartrate.
3. Reagent C: alkaline copper solution: Mix 50 mL of A and 1 mL of B prior to use.
4. Reagent D: Folin-Ciocalteau reagent – reflux gently for 10 h a mixture
consisting of 100 g sodium tungstate (Na2WoO4
2H2O), 25 g sodium molybdate
(Na2MoO4
2H2O), 700 mL water, 50 mL of 85% phosphoric acid, and 100 mL
of concentrated hydrochloric acid in a 1.5-L flask. Add 150 g lithium sulphate,
50 mL water and a few drops of bromine water. Boil the mixture for 15 min
without condenser to remove excess bromine. Cool, dilute to 1 L and filter. The
reagent should have no greenish tint. (Determine the acid concentration of the
reagent by titration with 1 N NaOH to a phenolphthalein end-point). Folin-
Ciocalteau reagent can be purchased commercially. Store refrigerated in amber
bottles. A good quality reagent is straw yellow in colour.
5. Protein solution (stock standard): weigh accurately 50 mg of bovine serum
albumin and dissolve in distilled water and make up to 50 mL in a standard flask.
6. Working standard: dilute 10 mL of the stock solution to 50 mL with distilled
water in a standard flask. One millilitre of the solution contains 200 mg protein.

Procedure
Extraction of protein from sample: Extraction is usually carried out with buffers
used for the enzyme assay. Weigh 500 mg of the sample and grind well with a pestle
and mortar in 5–10 mL of the buffer. Centrifuge and use the supernatant for protein
estimation.
Estimation of protein:
1. Pipette out 0.2, 0.4, 0.6, 0.8 and 1 mL of the working standard into a series of test
tubes.
2. Pipette out 0.1 and 0.2 mL of the sample extract in two other test tubes.
3. Make up the volume to 1 mL in all the test tubes. A tube with 1 mL of water
serves as the blank.
4. Add 5 mL of reagent C to each tube including the blank. Mix well and allow to
stand for 10 min.
5. Then add 0.5 mL of reagent D, mix well and incubate at room temp in the dark
for 30 min. Blue colour is developed.
6. Take the readings at 660 nm.
7. Draw a standard graph and calculate the amount of protein in the sample.
7.14 Polycrylamide-Sodium Dodecyl Sulphate Slab Gel Electrophoresis. . . 115

Calculation
Express the amount of protein mg/g or 100 g sample.
• If protein estimation is desired in a sample with high phenolic or pigment
content, extract should be prepared with a reducing agent preferably cysteine
and NaCl. Precipitate the protein with TCA, separate the protein and dissolve in
2N NaOH and proceed.
• If the protein concentration of the sample is high (above 50 mg/mL) measure the
colour intensity at 550 nm.
• For complete enzyme extraction, sometimes the chemicals like ethylenediamine
tetraacetic acid (EDTA), magnesium salts and mercaptoethanol are included.
This method of protein (mercaptoethanol) compounds as they interfere with this
procedure. When these chemicals are present in the extract, precipitate the
protein by adding 10% TCA, centrifuge and dissolve the precipitate in 2 N
NaOH and proceed for protein estimation.
• Rapid mixing as the Folin reagent is added is important for reproducibility.

7.14 Polycrylamide-Sodium Dodecyl Sulphate Slab

Gel Electrophoresis (SDS-PAGE) of Proteins
(Laemmli 1970)

Electrophoresis of proteins in polyacrylamide gels is carried out in buffer gels

(non-denaturing) as well as in sodium dodecyl sulphate (SDS) containing
(denaturing) gels. Polyacrylamide gel is more convenient than in any other medium
such as paper and starch gel. Electrophoretic procedures are rapid and relatively
sensitive requiring only micro-weights of proteins. Separation in buffer gels relies
on both the charge and size of the protein, whereas it depends only upon the size in
the SDS-gels. Analysis and comparison of proteins in a large number of samples is
easily made on polyacrylamide gel slabs.
Polyacrylamide gels are formed by polymerising acrylamide with cross-linking
agent (bisacrylamide) in the presence of a catalyst (persulphate ion) and chain
initiator (TEMED; N,N,N,N-tetramethylethylene diamine). Solutions are normally
degassed by evacuation prior to polymerization since oxygen inhibits polymeriza-
tion. The porosity of the gel is determined by the oxygen inhibited polymerization.
The porosity of the gel is determined by the relative proportion of acrylamide
monomer to bisacrylamide. Gels are usually referred to in terms of the total
percentage of acrylamide and bis present, and most protein separations are
performed using gels in the range 7–15%. A low percentage gel (with large pore
size) is used to separate high molecular weight proteins and vice versa. At high
concentrations of persulphate and TEMED the rate of polymerization is also high.
Among a number of methods commonly used, the sodium dodecyl sulphate-poly-
acrylamide gel electrophoresis (SDS-PAGE) that facilitates characterization of
polypeptides and determination of their molecular weight is described.