#36- Why is lane 3 a smear? The figure shows E1, E2, GST-ARDI (1-574), GST, and
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#36- Why is lane 3 a smear?
The figure shows E1, E2, GST-ARDI (1-574), GST, and GST-cytohesin-1 (C-1) added to proteins that can be ubiquitinated and run on a gel. The asterix denotes heat-inactivated protein. The samples were incubated with an anti-ubiquitin antibody E1 SDS-PAGE, westermy natve TE14 35, what proteins are 35. What proteins are required for ubiquitination in this experiment? 1-574 GST+ C-1 kDa OST ELE2 36, why is Lane 3 a smear? (Hint: similar to the Endo H question #16) 220- 37. Why were GST and C-1 added to Lanes 1 and 2? 90- 38. What was the purpose of the heat inactivation?Explanation / Answer
34. Western blot. The picture is added with anti-ubiquitin antibody to detect ubiquitination.
35. E1, E2, and GST- ARG1 (1-574)
36. The proteins E1, D2, and 1-574 made the proteins ubiquitinated. Anti-ubiquitin antibody is bound to ubiquitinated proteins in this lane. The chromogenic substrate of the antibody - enz complex gave the color that appers to be a smear
37. GST and C1 are added to see if they contribute to ubiquitination in the absence of 1-574
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