Chapter 5. 5.3 Immunoblotting

Assessments for Animated Techniques
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5.3 Immunoblotting

1.

Protein immunoblotting is also referred to as blotting.

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2.

A antibody recognizes a target of interest directly, whereas a antibody is designed to bind to the conserved region (Fc domain) of another antibody and is often conjugated to an enzyme or other molecular marker.

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3.

The step is required to bind nonspecific sites of the nitrocellulose membrane so that the primary and secondary antibodies will only bind specifically to their target molecules on the blot.

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4.

True or False: In SDS-PAGE, the proteins run towards the positive lead because they have been coated with the negatively charged SDS detergent molecules.

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B.

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5.

True or False: Detection of individual proteins using SDS-PAGE analysis alone is often possible.

A.
B.

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6.

True or False: Following the transfer step, the nitrocellulose membrane is an exact replica of the SDS-PAGE gel.

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B.

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7.

Click on the step and the associated process to identify the correct sequence of steps that are required to set up the transfer step of the immunoblotting process, once you have run out your SDS-PAGE gel. Note: In the diagram, the positive side of the apparatus is where you will assemble your transfer experiment.

Immunoblot with Power Supply
Step 1
Step 2
Place the SDS-PAGE gel.
Place the nitrocellulose membrane.
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8.

Click on the step and the associated process to identify the method steps required to complete the immunoblotting of the nitrocellulose membrane into their proper sequence.

Step 1
Step 2
Step 3
Step 4
Step 5
Step 6
Step 7
Step 8
Block the nonspecific protein binding sites on the nitrocellulose membrane, by incubating with serum albumin.
Incubate the membrane with the primary antibody of interest.
Crosslink the proteins transferred to the nitrocellulose membrane using UV light.
Wash the excess primary antibody from the blot.
Perform enzymatic reaction and detect signal.
Incubate the membrane with the enzyme-linked secondary antibody.
Remove the blocking agent from the membrane with a wash step.
Wash the excess secondary antibody from the blot.
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