Question

part c – order the steps (write 1–5)
____ add antibody
____ separate proteins by sds - page
____ detect signal with substrate
____ transfer proteins to membrane
____ block membrane

part d – application

1. a darker band appears for protein x in sample a than sample b. what does this mean?

Explanation:

Part C - Order the Steps

The Western Blot procedure (the process these steps belong to) has a standard order:

Step1: Separate proteins by SDS - PAGE

First, proteins are separated based on their molecular weight using SDS - PAGE.

Step2: Transfer proteins to membrane

After separation, the proteins are transferred from the gel to a membrane (like nitrocellulose or PVDF).

Step3: Block membrane

Blocking the membrane prevents non - specific binding of antibodies.

Step4: Add antibody

Then, the primary (and sometimes secondary) antibody is added to bind to the target protein.

Step5: Detect signal with substrate

Finally, a substrate is added to detect the signal from the antibody - protein complex.

So the order is:

• Separate proteins by SDS - PAGE: 1
• Transfer proteins to membrane: 2
• Block membrane: 3
• Add antibody: 4
• Detect signal with substrate: 5

In a Western Blot, the intensity of the band is related to the amount of the target protein. A darker band for Protein X in Sample A than in Sample B means that the amount of Protein X in Sample A is greater than the amount of Protein X in Sample B. This is because the antibody binds to Protein X, and the detection signal (which produces the band) is proportional to the quantity of the protein - antibody complex, and thus proportional to the amount of Protein X.

Answer:

1. Separate proteins by SDS - PAGE
2. Transfer proteins to membrane
3. Block membrane
4. Add antibody
5. Detect signal with substrate

Part D - Application