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ABSTRACT
Proteomics is a study of proteins in order to give a comprehensive view of
structure and functions to understand the biological process
INTRODUCTION
Protein is a large molecule composed of one or more chains of amino acids in
a specific order determined by the base sequence of nucleotides in the DNA coding
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4. Quaternary Structure
The structure formed by monomer monomer interaction in an oligometric
protein. It is the overall protein structure that results from the agregation of
these polypeptides subunits.
Human serum is devided in to two major group of protein which are albumin
and globolin. Albumin and globulin are filling out about 96 % of total serum proteins.
The normal amount of total serum protein in a healthy adult is about 55 90 mg/ml.
Total Serum protein can be interpreted as the total amount of protein that is found in
the blood.
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stands
for
sodium
dodecyl
sulfate-polyacrylamide
gel
electrophoresis. The SDS portion is an anionic detergent, meaning that dissolved its
molecules have a net negative charge within a wide PH range. The SDS has a
hydrophobic tail that interacts strongly with protein chains. Each SDS molecule
contributes two negative charges, overwhelming any charge the protein ay have.
SDS binds to all proteins and breaks up the weak
proteins, as such the protein will form one straight line. Protein separation by SDSPAGE can be used to estimate relative molecular mass, to determine the relative
abundance of major protein in a sample, and to determine the distribution of proteins
among fractions. There are two fractions which are Ep and Es. Ep fraction has more
protein than the Es fraction.
The purpose of ethanol precipitation is to reduce the solubility of protein.
Ethanol precipitation is one method for removing SDS and other alcohol soluble
impurities from protein samples with minimal protein loss. The protein lysate was
precipitated with ethanol and subjected to SDS PAGE with quantitative Coomassie
blue staining.
Glucose is our body main source of energy. This type of sugar comes from
digesing carbohydrates into chemical that we can easily convert to energy. We get
most of glucose from digesting sugarm and starch in carbohydrates. Blood sugar
level is controlled by some hormones such as insulin and glucagon. Insulin is a
hormone that control or regulate carbohydrate in the body, lowers the blood sugar
level. Insulin is produce in the beta pancreatic cells. Glucagon is also a hormone that
3
Ethanol Precipitation
Materials : Laemmli sample buffer, Coomassie blue staining solution ( 50%
methanol, 0.05% (v/v) Coomassie brilliant blur R-250, 10 % (v/v) Acetic Acid, 40%
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SDS-PAGE
Materials : Laemmli sample buffer, Coomassie blue staining solution ( 50%
methanol, 0.05% (v/v) Coomassie brilliant blur R-250, 10 % (v/v) Acetic Acid, 40%
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GEL STAINING
Materials : Laemmli sample buffer, Coomassie blue staining solution ( 50%
methanol, 0.05% (v/v) Coomassie brilliant blur R-250, 10 % (v/v) Acetic Acid, 40%
H2O ), TEMED
First the gel was removed and stained with coomasiie blue in wooble table for
1 hour. Then the gel was destained with destain solution in wooble table for 1 our,
the destaining solution was changed every 1 hour with fresh one until background is
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ELISA
Materials : Rabbit anti human AFP coated microtiter plate with 96 wells; zero
buffter, 13 ml; Reference standard set, contain 0, 5 , 30, 50, 250, 300 ng/ml AFP,
lyophilized; Enzyme Conjugate Reagent, 18 ml; TMB reagent, 11 ml; Stop soultion
(1N HCL0, 11) ml; Distilled water; Microtiter plate reader ( with a bandwidth of 10 nm
or less and an optical density range of 0-2 OD or greater at 450 nm wavelenght ).
First, the desired number of coated wells in the holder was secured. 20 ul of
standard, speciments, and controls was dispensed into appropriate wells. Then 100
ul of zero buffer was dispensed into each well. After that, it was mixed throughly for
30 seconds. Next, was incubated at room temperature for 30 minutes. The
incubation mixture was removed by flicking plate content into waste container. The
microtiter wells was rinsed and flicked 5 times with distilled water. Then the wells was
striked onto absorbent paper or paper towel to remove all residual water droplets.
150 ul of Enzyme Conjugate Reagent was dispensed into each well. Then was
gently mixed for 5 seconds. Next, it was incubated at room temperature for 30
minutes. The incubation mixture was removed by flicking plate content into a waste
container. The microtiter wells was then rinsed and flicked 5 times with distilled
water. The wells was striked sharply onto absorbnt paper to remove residual water
droplets. 100 ul of TMB reagent was dispensed into each well, was mixed for 5
seconds and then was incubated at room temperature for 20 minutes. The reaction
was stopped by adding 100 ul of Stop Solution to each well and was gently mixed for
30 seconds. Finally the optical density was read at 450 nm with a microtiter reader
within 15 minutes.
Concentration
Glucose standar
(mg/dl)
50
80
100
150
(mL)
0.6
0.8
1
1.2
1
2
3
4
Blank
Table 1 Concentration of Glucose Standard
Water (mL)
Total mL per
1.4
1.2
1
0.8
2
tube
2
2
2
2
2
2 mL 1% O-toluidine as added and mixed well. The tube as put in a boiling water
bath for 10 minutes. Finally, the absorbance was measured at 630 nm.
Measuring of blood glucose
Blood is drawn from a vein and transfered into a centrifuge tube. Next, serum
s obtained by centrifugation of blood for 10 minutes. Glucose concentration was
detemined in the provided serum sample using the O-toluidine method as follows :
0.1 ml of distilled water or standard glucose or serum was added in a clean dry test
tube. Then 2 ml of O-toluidine reagent was added.
Blank
Distilled water
0.1 ml
Standard
Test
O-toluidine
1 ml
Table 2 Volume of Standard and Test
Standard
50 ul
1 ml
Test
50 ul
1 ml
The content of each tube was mixed and the tube opening was covered with
aluminium foil. Then the tubes was put in a boiling water bath for 10 minutes. Test
tube was removued from the water bath and cooled under tap water. Next, the
absorbance was read at Imax 630 nm. Finally, the concentration of BSL in the
provided blood samples was calculated using the absorbance reading of standard
glucose.
RESULT
Standard curve of Bradford assay and the result of our sample calculation
Protein
Measurement
Absorbance
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at 595 nm
mL)
Y
1
2
X
0.125
0.142 0.143
0.1425
0.25
0.295 0.298
0.2965
0.5
0.483 0.488
0.4855
0.75
0.724 0.728
0.726
1
0.815 0.814
0.8145
Table 3 Concentration absorbance of BSA standard test (absorbance at 595
nm)
The absorbance at 595 nm of 5 samples of known concentration is recorder 2 times
each, and the average value is calculated.
0.8
0.7
0.6
0.5
Absorbance at 595 nm
Sample 1 (A)
Sample 2 (W)
1
0.532
0.673
2
0.539
0.678
Average (x)
0.5355
0.6755
ES
75
50
25
EP
EP
marker
MW
S1
75
S2
50
S3
25
S4
z
Table 5 Standard of SDS Page
log
4.875
4.699
4.398
x
d
1.25
1.9
3.4
4.5
L
5.6
5.6
5.6
5.6
Rf (d/L)
0.223
0.339
0.607
0.804
Standard of SDS-PAGE
0.7
0.6
0.5
0.4
Rf 0.3
0.2
0.1
0
4.3
4.4
4.5
4.6
4.7
4.8
4.9
Log
log = x
11
= antilog x
= antilog 4.143302958
= 13909.2271Da
= 13.9 kDa
Standard curve of ELISA test and the result of your sample calculation
A
0.041
0.086
0.156
0.388
0.800
S1
S2
S3
S4
S5
Table 6 Standard for ELISA
120
100
Concentration (ng/mL)
80
60
40
20
0
0
Standard curve of blood sugar determination assay and the result of our
sample calculation
A1
A2
S1
0.194
0.194
S2
0.315
0.318
S3
0.329
0.325
S4
0.428
0.43
Table 7 Standard for blood sugar determination
Mean A
0.194
0.317
0.327
0.429
Conc.
50
80
100
150
80
60
40
20
0
0.15
0.2
0.25
0.3
0.35
0.4
0.45
A2
0.313
0.300
Mean A :
-
Sample 1 = (0.313+0.313) 2
= 0.313
Sample 2 = (0.294+0.300) 2
= 0.297
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y = 423.17x 38985
y (sample 1) = 423.17(0.313) 38.985
= 132.45221 38.985
= 93.46721 mg/dL
y (sample 2) = 423.17(0.297) 38.985
= 125.6814 38.985
= 86.69649 mg/dL
DISCUSSION
The Bradford assay uses BSA ( Bovine Serum Albumin ) standard as a
comparison to determine the unknown serum protein concentration. This is because
of the close resemblance of the bovine protein to human blood which mostly consist
of albumin. In this experiment the absorbance reading is done 2 times to avoid the
erorr that can happened. The 2 result dont have a significant difference, this means
that the standard curve is accurate as basis for comparison.
On the calculation of the protein on sample A and B we already know that the
concentration of protein on sample A is 50.01 mg/ml, and the concentration on
sample B is 60.92 mg/ml. Based on the literature, the protein concentration on
sample A is a little bit low compared to the normal plasma concentration that is
between 50 90 mg/ml in a healthy adults, and normal protein concentration on
sample B. The result that happened in sample A can be happened due to errors in
the working method or lack of protein in the blood. The other reason that can cause
this low concentration of protein is the healthiness of the person. Usually, the protein
concentration in the blood can be decreased if the person is not in a healthy
condition or sick. Globulin is the building block for immunoglobulins, the main protein
of the immune system. With immune deficiency, the number of immunoglobulins is
reduced, which decreases blood protein levels.
Malnutrition, overhydration and affect of drugs can also cause low
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are normal, but the ratio of proteins to fluid has decreased. Several pharmaceuticals
can reduce total blood protein levels.
contraceptives and any drug that is toxic to the liver. Most error is because of human
eror. Improper mixing the dilluted serum protein immediate after injection of
coomassie stain.
In SDS Page, the more intense colouration of the band, the more abundant
that specific protein responsible for that band and the bigger molecular weight of the
protein, the more it will be retained. Ep fraction contains the precipitate proteins from
the result of ethanol precipitation. Es fraction contain protein that do not precipitate
after the ethanol precipitation procedure. These are the proteins found in the
supernatant.
Ep band travels further than the Es band determine by the distance they
travelled from loading the well. The small molecules of protein will migrate faster
than the larger molecules. SDS interaction with the protein cause the protein
migrates to the anode because of the overall negative charge.
As we can see in the result of SDS PAGE gel, there was a thick band around
50 molecular weight. The thick band can be interpreted that albumin is the major
protein blood plasma so it can be conjectured that the dark and heavy band in both
Ep and Es bands of both subjects are caused by the abundant albumin. The thick
bands means that there is same protein in both subjects. There is also possible that
the band is not in a straight line. This is happened because of error in preparing the
gel and the polymerization is not event in the gel. The different polymerization make
the result that same bands are bent line.
The aim of ELISA test is to determine of the Antigen AFP concentration in
human serum. ELISA test are utilized to detect substances that have antigenic
properties, primarily proteins. The concentration of AFP of sample A is -1.1442 ng/mL
and for sample B is -2.5176. Compared to the normal range of AFP concentration
that is <20 ng/mL the cocentration of AFP in sample A and B is normal. In some
cases the concentration of AFP can be elevated when hepatocellularcarcinoma or
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REFERENCE
http://www.kendricklabs.com/EtOHppt_2008.pdf
Glucose, Available at :
http://en.wikipedia.org/wiki/Glucose
Anonymous. Laboratory protocol for Fundamental Medical Science 1. Faculty
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