Organize the steps that take place for SDS-PAGE leading into Western Blotting:
Transfer proteins on gel to membrane
Denature protein sample
Load protein into gel
Treat protein with SDS
Apply electric current to gel
Apply antibodies
Photoimage luminescent proteins
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You learned that the NLS protein tag is both necessary and sufficient for nuclear localization of a protein. Let’s apply this concept to proteins that localize to the ER lumen. When answering the following questions, pick from the following:
Start transfer sequence
ER retention signal
Membrane Proteins
Explain how the following experiments can be used to determine the domain structure (or “topology”) of a membrane-associated protein in conjunction with SDS-PAGE.
a. Treatment with a protease.
b. Treatment with a detergent.
Practice: Detecting Proteins
You are working in a research lab studying a novel protein called WIZE. You have two versions of the protein, WIZE-14, which has incorporated light nitrogen, N14 and WIZE-15, which has incorporated heavy nitrogen, N15. Otherwise, they are exactly the same with the same folding structure and biological activity. One morning, you come into the lab and realize that you accidentally forgot to label your tubes last night. Whoops! What is the best technique to determine which tube has WIZE-14 and which tube has WIZE-15?
Which of the following methods requires the use of antibodies generated against the protein of interest?
You are studying a mutated form of Protein X, which has one missense mutation in it's DNA sequence. This mutation results in an amino acid substitution from an Ser to a Tyr. The mutated Protein X is folded into the same native conformation as the wildtype protein and has the same biological function. Which of the following is the best way to separate these two proteins?
How is chromatin immunoprecipitation (ChIP) different from regular immunoprecipitation (IP)?
The HES1 protein is a bHLH protein that is known to bind to certain gene regulatory motifs and repress transcription. You notice that when HES1 is mutated, the protein Ngn3 is up-regulated. How would you confirm that Hes1 associates with the Ngn3 transcript?
Which of the following protein purification methods can be used to isolate the phosphorylated form of a protein?
Use the following SDS-PAGE results for this 4 part question:
Use the following SDS-PAGE and Western Blot results for this 2 part question:
Practice: SDS-PAGE
You are working in a research lab studying a novel protein called WIZE. You have two versions of the protein: the standard version (30 kDa) and a version that has been post-translationally modified with a single ubiquitin molecule (which adds ~8.5 kDa to its mass). You accidentally forgot to label your tubes. What is the best and most direct technique to determine which tube contains the larger, ubiquitinated version of WIZE?