FINAL Q and A - Q May I please ask you a question regarding...

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Q: May I please ask you a question regarding the protocol for performing the colorimetric assay with alkaline phosphatase (AP)? I understand that AP will react with X-P. X-P will then react with NBT to make NBT-formazan. My question is during the lab, we added alkaline phosphatase buffer. My initial thinking was that the secondary antibody has the enzyme (alkaline phosphatase) already attached to it. Why do we need to add alkaline phosphatase buffer? A: The alkaline phosphatase enzyme is indeed already attached to the secondary antibody. You are adding the buffer to keep the enzyme happy (pH and such). That is why it is called alkaline phosphatase buffer. Q: I was talking with some former students and they mentioned that there  was one question that stumped them during the final they took last year.  They couldn't remember the entire question, but it was somewhere along the  lines of what were the three components of Km and talk about each of the  components. They said that they felt the question was unclear and that I  should ask about it so that I understand the concept better and not repeat  their same mistakes. Feel free to email me whenever you can. Thank you. A: I was talking about this question in lecture and in the review session. I had asked them to describe the 3 different components of Km (meaning the rate constants for ES consumption over ES formation. Furthermore, I had expected them to describe in this answer, what Km tells us: namely the affinity of an enzyme for its substrate and that it is the substrate concentration at which the enzyme velocity is half maximal. 1). Under Protein Purification, A. What makes the salt difference? On the slide it says that I-, CIO4- and few other salts are better as salting in, since they do not compete with water for the hydrophobic region of the protein. Is this what this question is referring? when the salt ions are smaller and have a smaller water shell themselves, they do not pull away the water from the proteins as effectively and hence the proteins stay longer soluble. B. I am not sure about a scenarios where it may be difficult to separate a certain protein from the bulk of other proteins by ammounium sulfate. Only thing I could assume would be a scenario in which protein can be extracted at a low ionic strength. when you test your specific protein (i.e. by an enzymatic assay at each step) and compare the relative activity (compared to the beginning) and that curve looks very much like the overall bulk proteins behave, then you won´t find a good concentration of ammonium sulfate, in which your protein is either more soluble than most proteins or less soluble than most proteins. In this case, it would be better to use another method of purification. And choosing a different salt won´t
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help here. I would go for a different property, such as size.
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