Lab 10 - Immunoblot

Question 1

Understand the benefits of this test (Immunoblot) compared to culturing techniques

Answer 1

• Quick turnaround time
• Cost effective
• Sensitive
• Specific
• Good if infectious agent is difficult to culture or dangerous to handle
• Results are either positive or negative; easy to interpret results

Question 2

Understand the 3 overall steps of an immunoblot, and the purpose of each step

Answer 2

Step 1: use gel electrophoresis to separate protein based on size (SDS-PAGE)
● Purpose: different proteins have different sizes. This is more important for step 3 when we need to know if we have the correct protein
Step 2: transfer proteins onto a nitrocellulose membrane
● Purpose: gel is very fragile, so we need something stronger to see if we have our protein of interest
Step 3: specially designed antibodies are used to identify a specific protein
● Purpose: this helps us see if we have our protein of interest since the antibodies are designed for solely detecting the protein of interest

Question 3

Know the purpose of an SDS-PAGE and what it stands for

Answer 3

Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis

It separates proteins based on molecular weight and conformation

Question 4

Know the difference between the stacking and resolving gels, and their purpose, in a normal discontinuous gel electrophoresis

Answer 4

Stacking gel:
- Located in the upper gel
- pH 6.8
- Acrylamide concentration 2-3%
- Purpose: makes resolved protein bands sharp and provides a better resolution
Resolving gel:
- Located in the lower gel
- pH 8.8
- Acrylamide concentration of 6-10%
- Purpose: separate proteins based on molecular weight and conformation

Question 5

Know the components of a sample buffer, and their purpose

Answer 5

SDS: denatures protein and gives it a uniform negative charge (proteins are now linear and can be separated solely by size)
Glycerol: sample density so it does not float out of the well when loading
β-mercaptoethanol: breaks down disulfide bonds
Bromophenol blue: tracking dye
Tris buffer at pH 6.8

Question 6

Know the different types of detection, and which was used specifically in this lab?

Answer 6

• Colorimetric detection (we used this one)
• Chemiluminescent detection
• Fluorescent detection
• Radioactive detection