Solved by a verified expert:Antibody Isolation
Practical write-up report
Student name:
Student number:
Introduction
Introduce what a polyclonal antibody is, and why and how you might want
to purify a particular immunoglobulin isotype away from the remaining serum
components
(maximum 300 words)
Methods
Present an extremely brief overview of the methods used
(maximum 200 words)
Results
There is no maximum word count
for any of the results section 1-5.
Section 1:
Present the results of your
ouchterlony and describe which column fractions were pooled to form fraction 5
(F5)
Section 2:
Calculate total protein of
all your fractions F1-5. Present all values as:
A. protein concentration in mg/ml
B. Total protein in your sample
having corrected your value for any dilution factor and the volume of the
sample
A calibration curve must be
completed and attached to this report. Sample values collected from the graph
must be clearly labeled
Section 3:
Calculate total IgG contained
within all of your fractions F1-5. Present all values as:
A. IgG concentration in mg/ml of
each sample
B. Total IgG in each sample having
corrected your value for any dilution factor and the volume of the sample
A calibration curve must be
completed and attached to this report. Sample values collected from the graph
must be clearly labeled
Section 4:
Calculate percentage yield of IgG
from the starting sample
((total IgG/ total protein) x
100)
Section 5:
Present the results of your immune-electrophoresis
clearly labeling where your samples were loaded onto the gel and which
antiserum was placed into which trough. Indicate where you observed lines of
precipitation.
Discussion:
Discuss the results from section 1-5 in terms of quantity yield and
quality of your samples. Critically review the relative advantages and
disadvantages of both purification methods
(maximum 300 words)ResultsResults for BSA (first week)
Concentration of
BSA (mg/ml)
Absorbance at 280nm
1.0
0.646
0.5
0.328
Results for Fractions( 1-4)
Fractions
Absorbance at 280nm
F1
0.621
F2
0.504
F3
0.242
F4
0.440
The fractions F1-F4 were diluted at 1/50Calibration curve were plotted from the BSA
absorbance and concentration in order to find the concentration of these
fractions 1-4. BSA and fractions 1-4 calibration curve
Fraction
Concentration mg/ml
F1
0.96
F2
0.78
F3
0.38
F4
0.68
Results for BSA (second week)
Concentration of BSA (mg/ml)
Absorbance @ 280nm
1
0.628
0.5
0.311
Fractions
Absorbance at 280nm
F5
0.340
F5 diluted at 1/50Calibration curve were plotted from the BSA
absorbance and fraction 5 absorbance from the second weekin order to find the concentration of fraction 5. BSA and
fractions 1-4 calibration curve
Fractions
Concentration mg/ml
F5
0.55
Section 1:Present the results of your ouchterlony and
describe which column fractions were pooled to form fraction 5 (F5)Ouchterlony Precipitation line was relatively visible for
samples 2, 3 and 5. These samples were pooled to make fraction 5.
6
2
3
5
4
Section 2:Calculate total protein of all your
fractions F1-5. Present all values as:A.
protein
concentration in mg/ml B.
Total
protein in your sample having corrected your value for any dilution factor and
the volume of the sampleA calibration curve must be completed and
attached to this report. Sample values collected from the graph must be clearly
labeledTotal
Protein = Concentration (mg/ml) x dilutions x volume F1: 0.96 x 50 x 0.5 = 24F2: 0.78 x 50 x 0.5 = 19.5F3: 0.38 x 50 x 0.5 = 9.5F4: 0.68 x 50 x 1= 34F5: 0.55 x 50 x 3 = 82.5
Fractions
Concentration
(mg/ml)
Dilution
Volume (ml)
Total concentration
(mg)
F1
0.96
50
0.5
24
F2
0.78
50
0.5
19.5
F3
0.38
50
0.5
9.5
F4
0.68
50
1.0
34
F5
0.55
50
3.0
82.5
Section 3:Calculate total IgG contained within all of
your fractions F1-5. Present all values as:A.
IgG
concentration in mg/ml of each sample B.
Total
IgG in each sample having corrected your value for any dilution factor and the
volume of the sampleA calibration curve must be completed and
attached to this report. Sample values collected from the graph must be clearly
labeled
1 0.5 0.2 0.1At dilution 1/20, fractions F1,
F2, F3, F5 circle formed. Fraction F4 didn’t form any circle.
Standards
Diameter
(mm)
Diameter(mm2)
1
13.32
177.4
0.5
11.21
125.7
0.2
8.90
79.21
0.1
7.11
50.55
Fractions
Diameter (mm)
Diameter (mm2)
F1
8.5
72.25
F2
8.8
77.44
F3
7.1
50.41
F4
0.0
0.00
F5
7.5
56.25
The diameter (mm2) for the
IgG standard were plotted on a graph and the diameter (mm2) for each
of the fractions that produced precipitation circles were plotted against the
line of best fit to determine the IgG concentration in each fraction. Single radial immunodiffusion of IgGCalculate total IgG contained within all of
your fractions F1-5. Present all values as: Following calculation was used to calculate the
total IgG in each fraction Total IgG concentration = concentration x
dilution x volumeF1: 0.20 x 20 x 0.5 = 2F2: 0.23 x 20 x 0.5 =2.3F3: 0.04 x 20 x 0.5 = 0.4 F4: didn’t form any circle.F5: 0.08
x 20x 3 = 4.8Concentration of IgG example of F1: look up
diameter of circle which for F1 is 72.25 and look across line of best fit and
go down which reads 0.20.
Fractions
Concentration of IgG
Dilution
Volume (ml)
Total IgG (mg)
F1
0.20
20
0.5
2.0
F2
0.23
20
0.5
2.3
F3
0.04
20
0.5
0.4
F4
0.00
0
0.0
0.0
F5
0.08
20
3.0
4.8
Section 4:Calculate percentage yield of IgG from the
starting sample((total IgG/ total protein) x 100)F1 1:20 (2/24) x 100 = 8.3%F2 1:20 (2.3/19.5) x 100 = 11.8% F3 1:20 (0.4/9.5) x 100 = 4.21% F5 1:100 (24/78) x 100 = 6.2% Section 5:Present the results of your immune-electrophoresis
clearly labeling where your samples were loaded onto the gel and which
antiserum was placed into which trough. Indicate where you observed lines of
precipitation.Plate 1Fraction three with a-IgG produced
a single precipitation line, indicating no other proteins present and pure IgG
is present. Fraction 1 with a-IgG produced multiple precipitation lines
indicating fraction 1 contained other proteins. Therefore fraction 1 was not
pure IgG. Moreover, fraction 2 with a-whs also produced multiple precipitation
lines. lines indicating fraction 1 contained other proteins. Therefore fraction
1 was not pure IgGPlate 2
Fraction 3 with a-whs showed
three precipitation lines so it’s not pure IgG. Fraction 4
showed no precipitin lines. Fraction 5 with a-whs showed many precipitation
lines indicating clearly other proteins were present therefore, fraction 5 was
relatively contaminated.Antibody Isolation 1
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