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heterok ary ons .
3. Warm fus ed het erok ary ons t o 37 degrees , f ix (c hemic ally k ill/ c ros s link and "f reez e" c ells ) s at
v arious t imes aft er warming
4. Immunos tain f us ed, f ix ed c ells wit h ant i mous e and ant i human antibodies
5. Vis ualiz e dis tribut ion of mous e and human c ell s urf ac e antibodies as a func t ion of t ime af ter
At 0 t ime, half the c ell has mous e membrane prot eins , half has human's . Stain c ell wit h
f luores c ent ant ibodies , s o half and half are s tained… af ter 40 minut es , both are t otally mix ed.
Why t emperature dependent ? At lower degrees , membrane is more rigid.
Ant ibody or lec t in induc ed pat c hing and c apping of c ell s urfac e prot eins demons t rat es induc ed
lat eral mov ement ; patc hing is ATP independent bec aus e all energy is built int o binding s it es ,
c apping requires ATP (t here is an underly ing c y tos k eleton and mot or proteins are try ing to driv e
t hem int o an aggregate).
Direc t v is ualiz ation of membrane protein mobilit y (or lac k t hereof ) by F R A P a n d F L I P
A nal y s i s : met hods f or f luores c ent ly t agging int egral membrane proteins :
a) monov alent fluores c ent lec t ins (nons pec if ic ) -- monov alent bec aus e more would driv e pat c hing
b) f luores c ent ly t agged monov alent (FAB f ragment s ) ant ibodies t o s pec ific protein
c ) t rans fec t c ells with c ons t ruc t enc oding (GFP-t agged membrane prot ein) and t hen
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This note was uploaded on 02/05/2014 for the course BIO 102 taught by Professor Chandhoke during the Fall '13 term at Yale.
- Fall '13