Explain the purpose of incubating the nitrocellulose

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1. Explain the purpose of incubating the nitrocellulose filter (NCF) in 5% (w/v) defatted milk powder in PBS/Tween before applying the primary antibody.

2. Could you use an alternative to the milk powder?

3. Describe how the western blot detection system utilizing an alkaline phosphatase conjugated secondary antibody and the substrate BCIP (5-bromo-4-chloro-indolyl-phosphate) works.

4. Another detection system available for western blotting involves enhanced chemiluminescence (ECL) what advantage does this offer compared to the AP-linked -BCIP method?

5. A researcher performs a routine western blot assay and is dismayed that there are no bands detectable, including the previously reliable positive control. She suspects that the transfer may have failed. Can you suggest a way of testing whether this is the problem?

6. Non-specificity is often a problem with western analysis especially when trying to detect a target protein in a complex mixture. Discuss some strategies to increase specificity.

Reference no: EM132598839

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