26 Jun An Investigation of ABO Group Antigen Structure The ABO blood group antigen epitopes are carbohydrates present on a cell membrane protein. The basic core carbohydrate (known as H) is present in group
uestion
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)
Results
Results 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 1/50
Calibration 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
.png”>.png”>.png”>.png”>.png”>.png”>
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 1/50
Calibration 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
.png”>.png”>.png”>
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.
.png” alt=”Text box: 1″>
.png”> .png”>
.png”>
.png”> .png”> .png”> .png”>
.png”> .png”>
6
2
.png”>.png”>
.png”> .png”> .png”> .png”>
.png”> .png”>
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 sample
A calibration curve must be completed and attached to this report. Sample values collected from the graph must be clearly labeled
Total Protein = Concentration (mg/ml) x dilutions x volume
F1: 0.96 x 50 x 0.5 = 24
F2: 0.78 x 50 x 0.5 = 19.5
F3: 0.38 x 50 x 0.5 = 9.5
F4: 0.68 x 50 x 1= 34
F5: 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 sample
A calibration curve must be completed and attached to this report. Sample values collected from the graph must be clearly labeled
.png”>.png”>.png”>.png”>.png”>.png”>.png”>
1 0.5 0.2 0.1
.png”>
.png”>
At 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 IgG
.png”>.png”>
Calculate 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 volume
F1: 0.20 x 20 x 0.5 = 2
F2: 0.23 x 20 x 0.5 =2.3
F3: 0.04 x 20 x 0.5 = 0.4
F4: didn’t form any circle.
F5: 0.08 x 20x 3 = 4.8
Concentration 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 1
Fraction 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 IgG
Plate 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
Our website has a team of professional writers who can help you write any of your homework. They will write your papers from scratch. We also have a team of editors just to make sure all papers are of HIGH QUALITY & PLAGIARISM FREE. To make an Order you only need to click Ask A Question and we will direct you to our Order Page at WriteDemy. Then fill Our Order Form with all your assignment instructions. Select your deadline and pay for your paper. You will get it few hours before your set deadline.
Fill in all the assignment paper details that are required in the order form with the standard information being the page count, deadline, academic level and type of paper. It is advisable to have this information at hand so that you can quickly fill in the necessary information needed in the form for the essay writer to be immediately assigned to your writing project. Make payment for the custom essay order to enable us to assign a suitable writer to your order. Payments are made through Paypal on a secured billing page. Finally, sit back and relax.
About Writedemy
We are a professional paper writing website. If you have searched a question and bumped into our website just know you are in the right place to get help in your coursework. We offer HIGH QUALITY & PLAGIARISM FREE Papers.
How It Works
To make an Order you only need to click on “Order Now” and we will direct you to our Order Page. Fill Our Order Form with all your assignment instructions. Select your deadline and pay for your paper. You will get it few hours before your set deadline.
Are there Discounts?
All new clients are eligible for 20% off in their first Order. Our payment method is safe and secure.