Weber et ans

Weber et ans - Weber et al The SNARE hypthesis is well...

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Weber et al The SNARE hypthesis is well accepted for explaining how vesicles identify the proper targets. What hypothesis about SNAREs is being tested in this paper? - Snare complex formation may also be sufficient to drive membrane fusion as well with targeting. Name the vSNARE and tSNAREs used in this paper. - VAMP2 and SNAP25 How was the v-SNARE purified? - Purified based on the binding property of his tag. The 6 histidines cause the protein to bind tightly and specifically to nickel. Agarose beads coated in nickel were incubated with the bacterial lysates and the VAMP. The beads were then washed and the VAMP was bound to them. The beads were then eluted with an excess of imidazole which also binds nickel and replaces the VAMP on the coated beads. from bacteria with C-terminal his tag on Ni-agarose beads - How were the t-SNAREs purified from bacteria?? Similar to the v-snares but the beads were coated with glutahione which binds GST. The complex was eluted by releasing the complex from the beads with a protease that cleaves between the GST and the VAMP portions of the polypeptide with N-terminal GST tag on only one of the proteins! Why do they only need a tag on one? What happened when they expressed each protein by itself? - ANSWER LATER How are the liposomes created? -
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Weber et ans - Weber et al The SNARE hypthesis is well...

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