This document describes an experiment using the Folin's phenol method to analyze total serum protein concentration. The key steps are: 1. A standard curve is generated by measuring the absorbance of known protein standards and plotting concentration versus absorbance. 2. The absorbance of a diluted serum sample is measured under the same conditions. 3. The protein concentration of the serum sample is determined by comparing its absorbance to the standard curve and adjusting for the dilution factor. The standard curve allows the protein concentration of an unknown sample to be determined based on its absorbance reading.

1. Biochemistry and molecular biology lab
Experiment 4
Folin’s phenol Method To
Analyze Total Serum Protein

2. Biochemistry and molecular biology lab
Clinical significance
The reference range for total protein is typically 60-80g/L. (It is
also sometimes reported as "6.0-8.0g/dl"), but this may vary
depending on the method of analysis.
Concentrations below the reference range usually reflect low
albumin concentration, for instance in liver disease or acute
infection. Rarely, low total protein may be a sign
of immunodeficiency.
Concentrations above the reference range are found
in paraproteinaemia, Hodgkin's lymphoma, leukaemia or any
condition causing an increase in immunoglobulins. Total protein
is also commonly elevated in dehydration.

3. Biochemistry and molecular biology lab
Purpose
 Learning Folin’s phenol method of
analyzing serum protein
 Practice standard curve determination

4. Biochemistry and molecular biology lab
Amino acid residues form a complex with
Cu2+ ion in basic medium

5. Biochemistry and molecular biology lab
 FFoolliinn’’ss pphheennooll mmeetthhoodd
Those protein containing phenol group can reduce
SStteepp 11
In alkaline
condition, Cu2+
form complex
with protein
The Cu-protein
complex reduce
molybdate.
phosphotungstic acid
and phosphomolybdate in blue color.
SStteepp 22
In blue color, the A value correlates with protein
concentration at 660nm.

6. Biochemistry and molecular biology lab
MMiinniimmaall aannaallyyzziinngg ccoonncceennttrraattiioonn iiss 55 μμgg//mmll,,
lliinneeaarr ddeetteeccttiioonn rraannggee iiss 2255--225500 μμgg//mmll ..
 AAddvvaannttaaggee::
 DDiissaaddvvaannttaaggee::
easy and reliable to operate
Protein specificity can influence
the result; as well as other
interfere substances.

7. Biochemistry and molecular biology lab
TTrriiss bbuuffffeerr gglluuccoossee
IInntteerrffeerree
ssuubbssttaanncceess
EEGGTTAA
AAmmmmoonniiaa ssuullffaattee
PPhheennoolliicc ccoommppoouunnddss
UUrreeaa
cciittrraattee
gguuaanniiddiinnee
ssooddiiuumm ssuullffaattee
TTCCAA
aallccoohhooll
aacceettoonnee
CCHHAAPPSS
EEDDTTAA
HHEEPPEESS
TTrriittoonn XX--110000
ddiitthhiiootthhrreeiittooll

8. Biochemistry and molecular biology lab
The reagent does not only measure phenols, and will
react with any reducing substance. It therefore measures
the total reducing capacity of a sample, not just phenolic
compounds. This reagent is part of the Lowry protein
assay, and will also react with some nitrogen-containing
compounds such as hydroxylamine and guanidine.
The reagent has also been shown to be reactive
towards thiols, many vitamins, the nucleotide base
guanine, the trioses glyceraldehyde and
dihydroxyacetone, and some inorganic ions. Copper
complex increases the reactivity of phenols towards this
reagent.

9. Biochemistry and molecular biology lab
Apparatus and reagents
 Large tube, measuring pipette, water bath;
 722E UV-vis spectrophotometer;
 Folin reagent (including reagent I and II);
 standard protein solution(250μg/ml);
 serum sample (diluted 500 times);

10. Biochemistry and molecular biology lab
In 7 large tube, Procedure adding reagents as following table
Blank
(1) (2) (3) (4) (5) (6)
Sample
（ 7）
Standard protein solution
(250μg/ml) -- 0.2 0.4 0.6 0.8 1.0 —
Serum sample — — — — — — 1.0
Distilled water 1.0 0.8 0.6 0.4 0.2 0 --
Folin reagent I 3.0 3.0 3.0 3.0 3.0 3.0 3.0
Mixture, and standing for 10min at room temperature.
Folin reagent II 0.3 0.3 0.3 0.3 0.3 0.3 0.3
Mix quickly !
Standing for 30min
Detecting absorption at 660nm, and obtain A2-A7.

11. Biochemistry and molecular biology lab
 Calculation and Drawing Standard
Curve
 SSttaannddaarrdd CCuurrvvee MMeetthhoodd//
CCaalliibbrraattiioonn CCuurrvvee
 SSttaannddaarrdd CCoonnttrrooll MMeetthhoodd

12. Biochemistry and molecular biology lab
In alkaline solution, pyruvate-dinitro phenyl
hydrazones emerge reddish-brown color with
the absorption peak in 510nm. The
absorbance value linearly correlates with
pyruvate concentration.
 lambert-beer law: A=ε c l
c：solution concentration
l：liquid thickness
A：substance absorbance
ε：molar absorbance coefficient

13. Biochemistry and molecular biology lab
Experiment results
Standard tube sample

14. Biochemistry and molecular biology lab
“MODE ”bottom：
setting detection mode;
“100%T”bottom：
setting 100.0%T
automatically;
“0%T”bottom：
setting 0.000%T
automatically;
“wavelength
setting”knob：
722S mode
722E mode
Use [MODE] button setting test manner: Transmittance (T), Absorbance (A),
Concentration (C).

15. Biochemistry and molecular biology lab
 SSttaannddaarrdd CCuurrvvee
A standard curve is a type of graph used as a quantitative research technique.
Multiple samples with known concentrations are measured and graphed, which
then allows the concentration of unknown samples to be determined by
interpolation on the graph. The absorbance value of each standard is then
determined by spectrophotometer. Plot a standard curve with the known
concentrations of protein, and the corresponding absorbance. The absorbance
value of a solution of unknown concentration is then determined under identical
conditions and the concentration can be simply read from the plot.

16. Biochemistry and molecular biology lab
Standard curve
＊
＊ ＊
＊ ＊
C
A
A6
A5
A4
A3
A2
0 C2 C3 C4 C5 C6
C7
A7
According to the standard plot, we can easily obtain C7 from
the curve.

17. Biochemistry and molecular biology lab
Calculation of protein concentration for the sample
(The serum has been diluted 500 fold)
SSaammppllee pprrootteeiinn
ccoonncceennttrraattiioonn==
CC77
（（gg//LL）） 11000000
××550000

18. Biochemistry and molecular biology lab
tube 1 2 3 4 5 6 7
OD660nm 0 0.121 0.250 0.335 0.440 0.525 0.320
MX
AX
MM77（（μμgg//mmll
））
××550000
11000000 CCsseerruumm==114499（（）） ==
μμgg//mmll
7744..55（（gg//LL））

19. Biochemistry and molecular biology lab
Notice
 at least 5 dot in a standard curve;
 standard solution and sample solution should be analyzed under the
same condition;
 the concentration of the solution should be in the linear range, and
absorbance value should be in the range of 0.05 ～1.0;

20. Biochemistry and molecular biology lab
Standard control method
Csample = Asample / Astandard× Cstandard
1. write all of those primary data;
2. calculation;
3. adding unit in concentration.

21. Biochemistry and molecular biology lab
Tube 1 2 3 4 5 6 7
OD660nm 0 0.121 0.250 0.335 0.440 0.525 0.320
CCsseerruumm==
00..332200
00..333355
××00..2255
VVssttaannddaarrdd
VVssaammppllee
00..66
11
AAssaammppllee
AAssttaannddaarrdd
××CCssttaannddaarrdd ××
××550000
== 7711..66 gg//LL
Normal range of human serum protein is
60-80g/L

22. Biochemistry and molecular biology lab
Assignment question
 What is standard curve？