In this experiment our intent was to detect the ligand binding of the serum albumen and phenol ruddy and detect it by utilizing the gel filtration chromatography.
Ligand is a substance, normally a little molecule that is able to adhere to a biomolecule. This binding produces a complex and serve as a biological intent ; activation, suppression or act as a neurotransmitter. It binds to a site of the mark protein. This binding of the ligand to protein occurs by intermolecular forces such as ionic bonds, H bonds or new wave der waals force.
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Because the binding is takes topographic point by intermolecular forces and non with covalent adhering which is a strong bond, ligand binding is reversible. Ligand binding changes the conformation of the receptor protein, the three dimensional form of the protein.
2.2 ) Serum albumen:
Serum albumen is a plasma protein in worlds and other mammals. This protein is of import for keeping the osmotic force per unit area which is needed for the distribution of organic structure fluids decently.
Albumin has six binding sites and it is soluble. It acts as a conveyance protein for hemin and fatty acids. It besides acts as a plasma bearer.
2.3 ) Gel filtration Chromatography:
gel filtration is a signifier of column chromatography technique that separates proteins, peptides and oligonucleotides on the footing of their size. In the column chromatography the stationary stage is solid and the nomadic stage is liquid. The stationary stage is put into a column which is normally a glass tubing and the nomadic stage that is a buffer or dissolver is allowed to flux through the solid stage. Here molecules will travel through a porous substance. This molecule while go throughing through will interact with the stationary stage and this interaction and the size of the molecule will consequence the clip that they leave the column. The grade of interaction between stationary stage and proteins depends on the belongingss of the protein, propeties of the stationary stage and the composing of the nomadic stage.
Figure 1. Conventional representation of separation by gel filtration chromatography.
3 ) EQUIPMENT AND CHEMICALS
3.1 ) equipments:
Chromatography column
Plastic cuvettes
Beakers
Clamps
Pipet
Tips
3.2 ) chemicals:
Distilled Water
BSA ( bovid serum albumen )
Acetate buffer
Phenol red
NaOH
Sephadex G25
4 ) Procedure
4.1 ) readying of the chromatography ( prepared earlier ) :
0.1 g phenol red was dissolved in 10 milliliter ethanoate buffer.
Chromatography was washed with buffer and so sephadex was poured into the column.
After gel was polymerized the gel was washed with buffer once more.
4.2 ) readying of the samples:
6 samples were prepared ( before the experiment ) with different concentration of phenol ruddy solution by blending the same ethanoate buffer and albumen. ( each group run 2 of the prepared samples in the chromatography. )
250 µl of the first sample was poured into the gel and so 50 milliliter of ethanoate buffer was added easy.
Samples that passed through the chromatography was collected in 40 cuvettes. In to the each cuvette merely 10 beads of the sample was added.
2 milliliter distilled H2O was added to the cuvettes.
200 µl 1 M NaOH was added to each cuvette.
Absorbance values of the all samples were measured at 520 nanometers.
stairss were repeated for the 2nd sample
5 ) CALCULATIONS/RESULTS
Table 5.1: optical density values and cuvette Numberss for sample 1 and 2 severally
0
1
0
2
0
3
0
4
0
5
0,004
6
0,009
7
0,013
8
0,014
9
0,024
10
0,034
11
0,038
12
0,047
13
0,071
14
0,086
15
0,133
16
0,157
17
0,234
18
0,278
19
0,243
20
0,244
21
0,228
22
0,207
23
0,167
24
0,16
25
0,127
26
0,089
27
0,062
28
0,044
29
0,04
30
0,02
31
0,015
32
0,007
33
0
34
0,002
35
0
36
0
37
0
38
0
39
0
40
1
0,017
2
0,013
3
0
4
0,008
5
0,013
6
0,037
7
0,067
8
0,028
9
0,056
10
0,017
11
0,032
12
0,037
13
0,037
14
0,076
15
0,083
16
0,104
17
0,16
18
0,228
19
0,325
20
0,461
21
0,512
22
0,635
23
0,694
24
0,749
25
0,803
26
0,7
27
0,674
28
0,575
29
0,454
30
0,389
31
0,311
32
0,198
33
0,146
34
0,11
35
0,083
36
0,052
37
0,037
38
0,019
39
0,018
40
0,031
41
0,007
42
0,01
43
0,009
44
0,001
45
0,013
46
0,011
absorbane vs. cuvette graph for sample 1.
For sample 2
Table 5.2: readying of the samples
Chemical reaction figure
Reagent
1
2
3
4
5
6
Bovine
serum albumen ( milligram )
20
20
20
20
20
20
Acetate buffer ( milliliter )
0.95
0.90
0.80
0.70
0.60
0.40
Phenol
ruddy solution ( milliliter )
0.05
0.10
0.20
0.30
0.40
0.606 ) Discussion
In this experiment our intent was to detect the ligand binding of the serum albumen and phenol ruddy and detect it by utilizing the gel filtration chromatography.
In this experiment we used albumin because different ligands can adhere to it easy. We used sephadex as gel in the experiment. Before utilizing it, it was waited in H2O for 3-4 hours so that it could swell.
During the experiment after roll uping 10 beads of the sample from chromatography we added NaOH and 2 milliliter H2O to the cuvette. The ground why we added NaOH was because we needed to obtain a coloured solution so that we could mensurate optical density values with spectrophotometer. In the acidic medium samples were xanthous. The ground why we added 2 ml H2O was besides because to be able to obtain optical density values. It is needed about 3 ml sample in the cuvette in order to be able to hold consequences.
Mobile stage was the ethanoate buffer and stationary stage was the sephadex gel. We were careful when we added our samples and buffer adding into the chromatography column. When we foremost poured the sample we added it truly easy and when we added buffer foremost we added a small sum of the H2O waited for it to travel in to the column and so we continued to add the buffer. We were careful when we added the buffer the solution which was at the top of the column non to travel up.
When we look at the optical density values we obtained from the sample 1 and 2 we see that they are different, because we used different sums of phenol ruddy and buffer in each sample. We were anticipating that in the samples after the chromatography there would be protein-phenol ruddy composite and phenol ruddy entirely. In the first cuvettes we would anticipate that protein sum would be higher because large molecules pass through chromatography, faster whereas little molecules go into porous gel and run slower in the column. Our graphs show that consequence but with some mistakes. Some optical density values were unexpected. It might be due to the incorrect handling of the cuvettes. Most of them were non clean, while making the experiment reagents that we used spread around it and besides bubbles were occurred.
We were anticipating that two extremums would happen in the graphs. We can detect that in the sample 2. They represent the serum albumin-phenol ruddy composite and merely the phenol ruddy.
we were able to detect what was in the cuvettes besides by the colour alteration in them. The first cuvettes that we filled were colourless than easy they became pink. Tap cuvettes were incorporating high sums of phenol ruddy and colourless 1s high protein.
