Introduction:
Hen egg white is one of the major raw materials used in food industry especially in
foaming and gelling. Its accessibility and numerous studies, however, lack the availability of
detailed knowledge and characterization of all the egg white component proteins. In order to
better describe egg white proteins, their functions, and identify the best purification methods,
various physico-chemical conditions are designed and applied (3). Egg white protein matrix is
difficult to analyze due to the protein molecular weight range (12.7-8000 kDa), isoelectric point
(pI) variation (3-10), enzymatic activity, and speciation (7). For example, ovalbumin represents
more than 50% of the total protein mass (w/v); hence, it impacts the detection of minor protein
species such as Avidin (7). As a result, the focus of this research project is the design of effective
purification methods for Avidin and method adjustment for Lysozyme (Lyz) separation from egg
white.
Lyz or N-acetylmuramide lycanhydrolase is a single polypeptide chain enzyme
containing 129 amino acid residues, with a molecular weight range of 14.3-14.6 kDa, and an
isoelectric point of 10.7. It represents approximately 3.5% (w/v) of the egg white (11). Avidin is
a dimeric or tetrameric non-enzymatic protein, with a molecular weight range of 33.0-35.0 kDa
in its dimeric form, and 66.0-69.0 kDa in its tetrameric form, and an isoelectric potential of 10.0.
Avidin represents approximately 0.05% (w/v) of the egg white and has a high-affinity for
binding biotin (6).
Lysozyme and Avidin isolation requires high-resolution protein separation methods. In
order to design high-yield purification techniques, protein thermal stability, molecular weights,
and isoelectric points are taken into account (12). Acidic pH of sample buffers isolates basic
protein species such as Lyz and Avidin from acidic and neutral ones (11). Thermal denaturation
eliminates thermally unstable contaminants. Chang (16) showed that the structural integrity and
activity of proteins decreases with the increase in time and temperature. For example,
immunoglobulin is thermo-sensitive, so that severe heat denaturation occurs at temperatures
above 75 ºC (20-22). Dialysis technique implies the loss of protein species with a molecular
weight (MW) smaller than the size of the semipermeable membrane pores. During dialysis, Lyz
and Avidin remain inside the semipermeable membrane (1). The MW and pI values variation,
electrophoresis procedure is applied to separate egg white proteins from each other (11).
Despite the fact that both Lyz and Avidin have been well characterized using Western
Blotting and ELISA, the procedures remain vague in details especially for Avidin quantitative
analysis from hen egg white protein samples (17-19). Besides, Avidin does not have enzymatic
functions and cannot be detected using enzymatic assays. Instead, it can be tracked by general
staining procedures and an over-loaded electrophoresis gel (18).
1|Page
This research is focused on two main hypotheses. The first hypothesis is the alteration of
acidic pH conditions of sample buffers and ultimate thermal denaturation will help optimize Lyz
and Avidin purification methods (5-12). The second hypothesis is that Western Blot and ELISA
procedures can be developed sensitive enough to detect small amounts of Avidin and Lyz.
General Objectives:
1. Optimization of Lysozyme and Avidin purification methods which include
sample buffer acidic pH alteration and heat denaturation.
2. Development of Western Blot procedure for Lysozyme and Avidin quantitative
analysis.
3. Development of ELISA procedure for Lysozyme and Avidin quantitative
analysis.
Research Project Design:
1. Optimization of Lysozyme and Avidin purification methods which include
sample buffer acidic pH alteration and heat denaturation.
a. Sample buffer pH conditions adjusted to pH of 4, 5, 6, and 7.
b. Heat denaturation of samples at 65ºC for 5 minutes.
2. Development of Western Blot procedure for Lysozyme and Avidin quantitative
analysis.
a. Western Blot procedure adapted for Lyz and Avidin concentrations.
3. Development of ELISA procedure for Lysozyme and Avidin quantitative
analysis.
a. ELISA procedure adapted to Lyz and Avidin concentration ranges.
Methods:
-Protein Preparation: Heat denaturation and isoelectric protein precipitation: After washing
and cleaning of eggs bought in trade, they are carefully broken and the white and yolk separated,
taking the precaution of removing chalazae (1). The separated egg whites are homogenized
using hand-held homogenizer. Avidin and Lysozyme were partially purified under acidic
conditions of pH 4.0, 5.0, 6.0, and 7.0. The volume of egg white (6 mL) is measured using a
graduated cylinder and then adjusted with 24 mL of ascorbate buffer solution (pH 4.0, pH 5.0);
and sodium phosphate buffer (pH 6.0, pH 7.0), with total buffer molarity of 50mM, at a 1:5 (v/v)
dilution ratio. The diluted samples were mixed via a gentle finger flicking for approximately 10
minutes and placed in a water bath for heat denaturation at 65°C for 5 minutes. The tubes were
immediately immersed in an ice bath for 1 minute to stop the denaturation (13-14). The pH of the
samples was measured after heat denaturation and readjusted to the initially set values using HCl
(2.0M) or NaOH (2.0M) as needed (Table #1).
2|Page
The tubes were centrifuged for 20 minutes in Beckman Refrigerated Centrifuge at 10,000 x g in
a JA 20.1 rotor. The pH of the centrifuged solutions was adjusted to pH 7.0 for ultimate tests
(Table#2). The crude samples were labeled as C1 and stored on ice.
-Ion-exchange Chromatography: Ion-exchange chromatography (IEC) technique was applied
on crude aliquots for Lyz and Avidin partial purification. IEC is based on the difference in
protein isoelectric potentials (pI) values eluting charged molecules from uncharged or in this
research project negatively changed and neutral protein species from positively charged protein
species (4). Lysozyme pI value is 10.7 and Avidin pI value is 10.0, which is greater than the pH
of the sample buffer; consequently, both proteins are considered to be positively charged and so
a cation exchange resin, S (Pierce) is used. In this experiment, the cation exchanger that is
negatively charged at pH value of 7.0 will electrostatically attract and bind proteins with a
positive charge at pH value of 7.0, like Lysozyme and Avidin, while the negatively charged or
electroneutral proteins will slip through the column.
The four aliquots of the prepared crude samples were exposed to dialysis as previously
described (8). The dialyzed samples were centrifuged in Beckman Refrigerated Centrifuge
(10,000 x g in a 20.1 JA rotor) for 20 minutes. Absorbance spectra were obtained from 1:20 and
1:40 dilutions using Beckman DU 800 Spectrophotometer at λ=280nm. The obtained samples
were eluted from the column with NaCl (150 mM) in sodium phosphate buffer, centrifuged in
Eppendorf Centrifuge (2000 x g JA rotor) for 5 minutes labeled as E1 and stored in ice for
ultimate tests. From the previously obtained absorbance values, protein concentration range was
estimated as 5µg-60µg and absorbance data were collected using Beckman DU 800
Spectrophotometer at λ=595nm. Linear graph was constructed and the molar extinction
coefficient was determined to be 0.0124.
-Bradford assay: The Bradford assay is a protein determination method that involves the binding
of Coomassie Brilliant Blue G-250 dye to proteins (2). When the dye binds to protein, it is
converted to a stable unprotonated blue form detected at λ = 595 nm (10). Beer's law may be
applied for accurate quantitation of protein by selecting an appropriate ratio of dye volume to
sample concentration. In this assay, E1 samples at estimated concentrations of 0.0958 mg/ml
(pH=7), 0.0617 mg/ml (pH=6), 0.0523 mg/ml (pH=5), and 0.0504 mg/ml (pH=4); and C1
samples at estimated concentrations of 0.253 mg/ml (pH=7), 0.239 mg/ml (pH=6), 0.246 mg/ml
(pH=5), and 0.275 mg/ml (pH=4) were tested (2). The absorbance data were obtained using
Beckman DU 800 Spectrophotometer at λ=595nm.
-Western Blotting: SDS-PAGE (Sodium Dodecy Laemmlil Sulfate Polyacrylamide Gel
Electrophoresis): Gels, buffers, and Coomassie Blue Stain were prepared according to the SDSPAGE Protocol. 15% acrylamide Criterion gels (BioRad) were used (See protocol SDS-PAGE
table). Sample loading concentrations were adjusted according to the results obtained from
Bradford assay.
3|Page
-Blotting: The “transfer sandwich” was run at 100V for 1 hour. The samples were blocked with
0.2% blocker Tween-20 solution and incubated with Avidin and Lysozyme primary antibodies
diluted at 1:2000 for 1 hour with shaking at 4ºC. The sequential incubation of primary and
antibodies is followed by brief washing of the blot with PBS-T (0.1%). Secondary antibodies
800CW (1:10,000) and 680LT (1:20,000) were added to the tested samples, and incubated for 1
hour at room temperature with shaking. The samples were imaged on LICOR el Imager at
700nm and 800nm for 15sec-5min.
-ELISA: Buffers and coating solutions were prepared according to the standard protocol. The
wells were filled with 100µL/well of coating solution, incubated overnight at 23ºC, washed 4x
with 350 µL PBS-T buffer per well, and blocked with 150µL/well blocking solution. 100
µL/well of primary antibody (80ng/mL antibody in PBS-T) were added, and incubated for 1 hour
at 23ºC. The plate was washed as previously stated and incubated for 1 hour at 23 ºC with 100
µL/well of second antibody (300ng/mL in PBS-T). The plates were washed and incubated with
100 µL/well of 1/3 TMB to citric acid (0.1M)/acetate (0.1M) solution adjusted at pH=4.9 for 20
minutes at RT. Calorimetric reaction was stopped by the addition of 100 µL/well 0.5M sulfuric
acid. Plates were screened in BioRad iMark Microplate Reader at λ=450nm.
Results and Discussion:
-Optimization of Lysozyme and Avidin purification methods: Lysozyme and Avidin
purification methods were optimized by the application of the sample buffers with pH values of
4.0, 5.0, 6.0, and 7.0. 1:5 (v/v) diluted samples were exposed to heat denaturation at 65°C for 5
minutes with pH readjustment (Table#1).
Table#1: Protein samples pH adjustment after heat denaturation.
pH=4
pH=5
pH=6
pH=7
initial
3.98
4.77
5.89
6.87
Final (after adjustment)
3.98
5.03
5.98
6.99
Heat denatured samples had different degree of cloudiness and precipitate formation. Egg
white samples at pH 7.0 and 6.0 were slightly cloudy whereas pH 4.0 had no sign of precipitate
formation. Samples exposed to pH 5.0 had the most amount of precipitate formation which might
be explained by the big number of proteins that fall in this range of pI 5.0 values.
The samples were centrifuged for 20 minutes in Beckman Refrigerated Centrifuge at
10,000 x g in a JA 20.1 rotor and the pH of the supernatant liquid was adjusted to pH 7.0 for
ultimate tests (Table#2).
4|Page
Table#2: Centrifuged protein samples’ pH adjustment.
pH=4
4.68
6.99
initial
final
pH=5
7.45
6.98
pH=6
6.50
6.99
pH=7
7.21
7.01
During the ion-exchange chromatography, the positive charge of Lyz and Avidin at pH
7.0 was used to eliminate electro-neutral and negatively charged protein species. Cation
exchange resin, S (Pierce) bound positively charged proteins like Lysozyme and Avidin, while
allowing the negatively charged or electro-neutral proteins slip through the column. The obtained
samples were exposed to dialysis as previously described (8). Absorbance data were obtained
from 1:20 and 1:40 dilutions using Beckman DU 800 Spectrophotometer at λ=280nm (Table#3).
Table#3: Absorbance at λ=280nm of 1/20 and 1/40 diluted protein samples:
1/20 dilution
pH=4.0
pH=5.0
pH=6.0
pH=7.0
1.171
0.911
0.996
1.040
1/40 dilution
pH=4.0
pH=5.0
pH=6.0
pH=7.0
0.549
0.491
0.477
0.506
Estimated
[protein] 1/40
dilution (mg/mL)
(E1)
0.0504
0.0523
0.0617
0.0958
Estimated
[protein] 1/40
dilution
(mg/mL) (C1)
0.275
0.246
0.239
0.253
Original
[protein] 1/40
dilution
(mg/mL)
11.0
9.82
9.54
10.1
The crude samples were washed with 50mM sodium phosphate, centrifuged in Beckman
Refrigerated Centrifuge (20.1 JA force) for 5 minutes, eluted, and labeled as E1. The protein
concentration range was estimated as 5µg-60µg and absorbance data were collected using
Beckman DU 800 Spectrophotometer at λ=595nm (Table#4).
Table#4: Absorbance at λ=595nm of protein samples at 5µg-60µg concentration range.
Protein Concentration (µg)
5
10
15
20
30
40
50
60
Abs
0.118
0.205
0.254
0.335
0.436
0.552
0.677
0.789
Abs
0.088
0.158
0.2
0.265
0.374
5|Page
Fig.1: Absorbance at λ=595nm of protein samples at 5µg-60µg concentration range.
0.9
0.8
y = 0.0124x + 0.0456
R² = 0.9852
0.7
0.6
0.5
Series1
0.4
Linear (Series1)
0.3
0.2
0.1
0
0
10
20
30
40
50
60
70
From Fig.1 the extinction coefficient was obtained from the equation: y=0.0124X+0.0456.
Using the obtained Beer Lambert equation, Table#5 was designed for the Bradford assay:
Table#5: Bradford assay design for E1 and C1 protein samples.
Id
E1(7)
E1(7)
E1(6)
E1(6)
E1(5)
E1(5)
E1(4)
E1(4)
C(7)
C(7)
C(6)
C(6)
C(5)
C(5)
C(4)
C(4)
V(µl)
100
100
100
100
100
100
100
100
12.4
12.4
13.1
13.1
12.8
12.8
11.4
11.4
Abs λ=595nm
0.240
0.253
0.196
0.197
0.136
0.142
0.144
0.152
0.460
0.495
0.435
0.458
0.350
0.373
0.307
0.287
m (µg)
15.68
16.73
12.13
12.21
7.29
7.77
7.94
8.58
33.42
36.24
31.40
33.26
24.55
26.40
21.08
19.47
µg/µl
0.1568
0.1673
0.1213
0.1221
0.0729
0.0777
0.0794
0.0858
2.6951
2.9227
2.3972
2.5388
1.9178
2.0628
1.8492
1.7077
average (µg/µl)
0.16
undiluted crudes(µg/µl)
0.12
0.08
0.08
2.81
14.04
2.47
12.34
1.99
9.95
1.78
8.89
6|Page
-Western Blot: SDS-PAGE (Sodium Dodecy Laemmlil Sulfate Polyacrylamide Gel
Electrophoresis) procedure optimization: Gels, buffers, and Coomassie Blue Stain were
prepared according to the SDS-PAGE Protocol. 15% acrylamide Criterion gels (BioRad) were
used (SDS-PAGE protocol table). Sample loading concentrations were adjusted according to the
results obtained from Bradford assay (Table#6).
Table#6: SDS-PAGE table before adjustment:
Id and Molecular weight
Standard
Avidin (68.3 kDa)
Lysozyme (14.3 kDa)
E1(7)
E1(6)
E1(5)
E1(4)
C1(7)
C1(6)
C1(5)
C1(4)
H2O (µL)
14
15
15
0
0
0
0
23.5
22.5
21.0
20.0
Sample (µL)
6
15
15
30
30
30
30
6.43
7.50
9.00
10.00
2xLammeli buffer (mL)
20
30
30
30
30
30
30
30
30
30
30
The wells were loaded according to the schemas:
SDS-PAGE schema for Avidin:
1
sb
2
3
Avidin E1(7)
4
5
6
7
8
E1(6) E1(5) E1(4)
9
C1(7)
10
C1(6)
11
12
C1(5) C1(4)
SDS-PAGE schema for Lysozyme:
1
sb
2
3
4
Avidin Lysozyme E1(7)
5
E1(6)
6
7
8
E1(5) E1(4) C1(7)
9
C1(6)
10
C1(5)
11
12
C1(4) ----
The “transfer sandwich” was run at 100V for 1 hour. The samples were blocked with 0.2%
blocker Tween-20 solution and incubated with Avidin and Lysozyme primary antibodies diluted
at 1:2000 for 1 hour with shaking at 4ºC. The sequential incubation of primary and antibodies is
followed by brief washing of the blot with PBS-T (0.1%). Secondary antibodies 800CW
(1:10,000) and 680LT (1:20.000) were added to the tested samples, and incubated for 1 hour at
room temperature with shaking. The samples were imaged on LICOR el Imager at 700nm and
800nm for 15sec-5min (Fig.2, Fig.3).
7|Page
Fig.2: SDS-PAGE Lysozyme (gel#2)
Fig.3: SDS-PAGE Avidin (gel#1)
Table #8: Avidin WB gel#1 Results:
E1(4)
ND
E1(5)
ND
E1(6)
ND
E1(7)
ND
C1(4)
24.8
C1(5)
43.7
C1(6)
34.9
C1(7)
ND
Signal-Avidin
SignalND
ND
ND
ND
ND
ND
ND
ND
Conalbumin
From Table #8, Avidin was detected with Western Blot only in C1 samples. The protein
was not detected most of the times because of the overload of the wells. Molecular Weight
detected was ~ 15.7kDa, monomeric structure compared to the dimer in the standard; maximal
recovery of ~2 fold with respect to the lowest recovery values from other buffers, was obtained
in ascorbate buffer at a pH of 5.0. Conalbumin was not detected in any of the wells. Lysozyme
was detected in C1(6), C1(5), and C1(4), which means that the used antibody was impure
affecting the quantitation.
Table #9: Lysozyme WB gel#2 Results:
SignalLysozyme
SignalConalbumin
E1(4)
E1(5)
E1(6)
E1(7)
C1(4)
C1(5)
C1(6)
C1(7)
4.82
10.8
3.33
5.20
85.1
80.3
93.5
76.8
ND
ND
ND
ND
ND
ND
ND
ND
From Table #9, Lysozyme was detected with Western Blot even in the eluted E1 samples.
Molecular Weight detected was ~ 14.9kDa; maximal recovery of ~3 fold with respect to the
lowest recovery values from other buffers was obtained in ascorbate buffer at a pH of 5.0.
Conalbumin was not detected in any of the wells. The antibody used in the experiment did not
interfere with Lysozyme quantitation.
8|Page
To avoid the overload of the wells, the protein volumes loaded were adjusted (Table#10).
Table#10: SDS-PAGE table after adjustment:
ID
Sb
Avidin
E1(7)
E1(6)
E1(5)
E1(4)
H2O(µL) Buffer NaCl (150mM), Na3PO4 (µL)
8.00
0
14.10
0
14.10
8.49
14.10
6.06
14.10
0
14.10
1.40
Sample (µL)
12.00
15.90
7.41
9.84
15.90
14.50
2xLammeli (µL)
20
30
30
30
30
30
The wells were loaded according to the schema:
1
sb
2
E1(7)
3
E1(6)
4
E1(5)
5
E1(4)
6
---
7
8
Avidin E1(4)
9
E1(5)
10
E1(6)
11
E1(7)
12
sb
The “transfer sandwich” was run at 100V for 1 hour. The samples were blocked with
0.2% blocker Tween-20 solution and incubated with Avidin and Lysozyme primary antibodies
diluted at 1:2000 for 1 hour with shaking at 4ºC. The sequential incubation of primary and
antibodies is followed by brief washing of the blot with PBS-T (0.1%). Secondary antibodies
800CW (1:10,000) and 680LT (1:20.000) were added to the tested samples, and incubated for 1
hour at room temperature with shaking. The samples were imaged on LICOR el Imager at
700nm and 800nm for 15sec-5min (Fig.4).
Fig.4: SDS-PAGE Lysozyme and Avidin (gel#3)
9|Page
Table #11: Lysozyme WB gel#3 Results:
Signal-lysozyme
SignalConalbumin
E1(4)
.635
E1(5)
2.08
E1(6)
.367
E1(7)
.460
.660
NaN
NaN
.338
From Table #11, Lysozyme was detected with Western Blot even in the eluted E1
samples. Molecular Weight detected was ~ 13kDa, maximal recovery of 5-6 fold with respect to
the lowest recovery values from other buffers, was obtained in ascorbate buffer at a pH of 5.0.
The pH conditions at 5 and 6 were the best for the removal of Conalbumin (Ovotransferrin). The
antibody used in the experiment was non-specific and did not interfere with Lysozyme
quantitation.
Table #12: Avidin WB gel#2 Results:
Signal-avidin monomer
Signal-Conalbumin
E1(4)
1.70
4.60
E1(5)
1.95
.722
E1(6)
.600
.739
E1(7)
.704
3.54
From Table# 12, Avidin was detected from the eluted E1 samples by overloading the
wells. The detected molecular weight was ~15.9 kDa, dimeric from; maximal recovery of ~3 fold
with respect to other buffer conditions was obtained in ascorbate buffer at a pH of 5.0. The pH
conditions at 5 and 6 were the best for the removal of Ovotransferrin. The used antibody was
non-specific and did not interfere with Avidin quantitative analysis.
-ELISA optimization: Enzyme-linked immunosorbent assay was designed to capture Lysozyme
only as an alternative detection method; Avidin should not be detected because it does not have
enzymatic activity. Buffers and coating solutions were prepared according to the standard
protocol. The wells were filled with 100µL/well of solution according to optimization calculation
(Table#13, 14, 15, 16).
Table#13: ELISA micro-plate dilution optimization.
E1(7)
E1(6)
E1(5)
E1(4)
Avidin
Lysozyme
3xblank
Sb
H2O(µL)
106.5
100.4
85
88.9
2xPBS(µL)
125
125
125
125
125
125
Sample(µL)
18.5
24.6
40
36.1
[Stock]ng/µL
162
122
75
83
0.1
V(total)(µL)
250
250
250
250
250
250
250
250
Dilutions
1:200
1:200
1:200
1:200
10 | P a g e
*Avidin and Lysozyme 1:10,000 dilution---- 1µg/µL= 0.1ng
Table#14: E1(5) micro-plate calculations.
ddH2O(µL)
118.3
105.0
85.0
E1(5),A
E1(5),B
E1(5),C
2xPBS(µL)
125
125
125
Diluted sample (o.375ng/µL) (µL)
6.7
20
40
Amount (ng)
2.5
7.5
15
Diluted sample (1.50ng/µL) (µL)
1.7
5.0
10.0
Amount (ng)
2.5
7.5
15
Table#15: E2(5) micro-plate calculations.
ddH2O(µL)
123.3
120.0
115.0
E2(5),A
E2(5),B
E2(5),C
2xPBS(µL)
125
125
125
Table#16: Standard solution calculation adapted for micro-plate screening.
ddH2O(µL)
120
115
110
95
Sb
Sb
Sb
Sb
2xPBS(µL)
125
125
125
125
Diluted sample (µL)
5
10
15
30
Amount (ng)
0.5
1
1.5
3
Micro-plates were screened in BioRad iMark Microplate Reader at λ=450nm following loading
map from Table#17.
Table#17: ELISA micro-plate loading map:
1
2
3
4
Blnk
Blnk
A Blnk
B LyzA LyzA LyzB LyzB
E1 B
C E1 A E1 A E1 B
D
E
F Av A Av A Av B Av B
E1 B
G E1 A E1 A E1 B
H
*Grey color- Lysozyme present.
5
6
LyzC
E1 C
LyzC
E1 C
Av C
Av C
E1 C
E1 C
7
8
9
10
11
12
Blnk
Blnk
Blnk
E2 A
E2 A
E2 B
E2 B
E2 C
E2 C
E2 A
E2 A
E2 B
E2 B
E2 C
E2 C
*A=[E1(5)]=2.5 ng; B= [E1(5)]=7.5 ng; C=[E1(5)]=15.0 ng;
*E2=4x[E1(5)]
11 | P a g e
Table #18: ELISA micro-plate Results:
Signal-Lyz
added
Signal-no
Lyz added
E1(5),
2.5ng
E1(5),7.5
ng
E1(5),
15.0ng
E2(5),
2.5ng
0.1165
0.0635
0.0585
0.060
0.0705
0.0685
0.0605
0.050
0.551
0.378
0.395
0.020
0.383
0.140
0.367
0.018
E2(5),7.5
ng
0.060
E2(5), 15.0ng
0.070
0.014
0.10
0.020
0.027
0.018
0.090
From Table# 18, Lysozyme was detected from the eluted E1 and E2(4x[E1]) samples. E2
samples showed poor detection due to the overload of the micro-plate wells. Maximal detection
of ~2 fold with respect to other E1(5) concentrations is acquired in E1(5), 2.5ng samples. The
signal is recorded even in the wells with no Lysozyme added with maximal detection in E1(5),
2.5ng samples. The used antibody did not show a good binding to enzymes.
Conclusions:
Egg white protein characteristics remain to be under-described due to the low
effectiveness of applied purification methods. In this research project, we analyzed one
enzymatic egg white protein- Lysozyme, and one minor non-enzymatic protein-Avidin. The two
minor species were tested for purity via buffer pH variation and heat denaturation at 65ºC.
Western Blot and ELISA were analyzed for Lysozyme and Avidin detection sensitivity.
Lowering the buffer pH to 5.0 caused Lysozyme and Avidin maximal purity and recovery. The
detected Avidin traces’ showed molecular weight of its dimer isoform. Heat denaturation at
65ºC did not facilitate protein purification. Western Blot detection method was remarked to be
more useful analytical technique for Lysozyme and Avidin purity estimation than ELISA.
Western Blot gel screenings allowed simultaneous visualization of eluted and crude samples.
ELISA is limited to Lysozyme analysis and was prone to speciation interference. Future research
might focus on the purification method optimization with higher heat denaturation temperature
ranges. Specific antibodies might be tested with Lysozyme and Avidin ELISA. Besides,
fluorescence assay might be introduced as a tool for more accurate protein purity estimation.
These future researches will lead to more specific quantitative protein tests and possibly the
design of better purification methods.
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