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Surface biotinylation and subsequent chase-to assess the surface protein turnove


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#1 GNANA

GNANA

    GNANA

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Posted 29 May 2012 - 08:00 AM

Hi,

I am planning to do a surface biotinylation experiment inorder to assess the half life/turn over of a receptor.

my experimental procedure:
Grow cells to around 70% confluent
on the day of biotin labelling-wash with ice cold PBS (+ca & mg)
label with biotin (1mg/ml) about 20 mins in PBS (on ice). (boitin purchased from pierce - Ez link NHS-ss-biotin , not the whole kit only biotin).
washes with PBS+100mM glycine (to quench unreacted biotin)
add prewarmed complete medium
Chase (1hr to 12 hrs)
lyse cells and IP down with Streptavidin
Western blot.


Anyone who experienced on this give your suggestion whether my protocol looks fine or shd i hav to do any modifications ? also i need to know whether i can freeze the cells after every chase......

Thanks in advance for your inputs.
Gnana.....
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....




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