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PEX-12-04

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29 views4 pages

PEX-12-04

Uploaded by

Mirey
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Exercise 12: Serological Testing: Activity 4: Western Blotting Technique Lab Report

Pre-lab Quiz Results


You scored 100% by answering 4 out of 4 questions correctly.

1. Which of the following tests is not correctly matched to what it tests for?
You correctly answered: d. These are all correctly matched.

2. Gel electrophoresis is used to separate proteins on the basis of


You correctly answered: e. size and charge

3. Western blotting can be used to test for


You correctly answered: d. HIV and Lyme disease

4. In this activity you will use Western blotting to test for the presence of
You correctly answered: b. antibodies (like the indirect ELISA).

010/28/18 page 1
Experiment Results
Stop & Think Questions:
What is present on the nitrocellulose strips?
You correctly answered: a. HIV antigens separated by electrophoresis

How would you describe the binding site of the secondary antibody?
You correctly answered: c. the constant region of the primary antibody

Experiment Data:

gp160 gp120 p55 p31 p24 HIV Test Result


Patient A no no no no no Negative
Patient B no no yes no yes Indeterminate
Patient C yes yes yes no yes Positive
Positive Control yes yes yes yes yes Positive
Negative Control no no no no no Negative

010/28/18 page 2
Post-lab Quiz Results
You scored 100% by answering 4 out of 4 questions correctly.

1. The patient samples being tested contain


You correctly answered: b. serum.

2. The primary antibodies in the Western blot


You correctly answered: c. come from the patient sample.

3. A key difference between the ELISA and the Western blot technique is that the
You correctly answered: a. Western blot looks at separated antigens.

4. The antibodies in a positive patient sample bind to the


You correctly answered: d. constant region of the enzyme-linked antibody and the antigen bound to the nitrocellulose strip.

010/28/18 page 3
Review Sheet Results
1. Describe why the HIV Western blot is a more specific test than the indirect ELISA for HIV.
Your answer:
The HIV Western blot has a discrete protrein band that represents the specific antigen the antibody is recognizing, while the
ELISA uses a well that corresponds to a mixture of antigens.

2. Explain the procedure for a patient with an indeterminate HIV Western blot result.
Your answer:
Los pacientes que tras hacer el experimento se consideran indeterminados, hay que repetirles las pruebas de nuevo en
una fecha posterior. Si las bandas están presentes pero no coinciden conlos criterios para un resultado positivo, se
considerará que son indeterminados. Para que los resultados sean positivos, debe haber bandas en GP160, GP120 y P31
o P24

3. Briefly describe how the nitrocellulose strips were prepared before reacting them with the patient samples.
Your answer:
La corriente eléctrica se utiliza para separar proteínas en función de su tamaño y carga. Esta técnica utiliza electroforesis
en gel para separar las proteínas en una matriz de gel, luego las proteínas se transfieren a una membrana de
mitrocelulosa.

4. Describe the importance of the washing steps in the procedure.


Your answer:
Los pasos de lavado eliminan cualquier unión no específica de anticuerpos conjugados secundarios para que no
reaccionen incorrectamente con el sustrato para dar un falso positivo.

010/28/18 page 4

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