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Autophagy detection problems


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#1 Pufffetta

Pufffetta

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Posted 13 August 2011 - 12:54 PM

Hello!
I'm working on autophagy since a couple of months, trying to get well results with LC3 Antibody.
I used two different antibodies, but none seems to work: I just can see aspecific bands (around 39 and 60-79 kDa) but I can't see the normal bands 17-19kDa.
I try to explain better what I'm doing...if you have any suggestion I will very appreciate.

I Lyse the LCLs cells in 1xRIPA+PMSF+DTT+Protease inhibitor cocktail and PhosSTOP, incubate 4C for 30 min; spin samples and collect supernatant.
Quantified proteins with Bradford reagent seems to give good results.
I use a 14% or a precasted 4-15% gel. I used load 30-60ug of sample per lane. Samples were prepared adding Laemmli buffer (2X) +5% 2-ME,then boiling samples at 95 for 5 min.
Electrophoresis is made 1hr 160V, the samples don't leave the gel.
To transfer:
I activate PVDF 0,22 membrane with methanol for 3min and then 30 min in transfer buffer (towbin buffer with 20% methanol-no SDS-). I also equilibrate the gel in transfer buffer for 20-30 min.
I used semidry transfer (16V for 1hr) or wet transfer (150mA for 1hr).
Immunoblot:
Blocking solution: I try with TBS-T 0.05% +5% NFDM and also with TBS-T 0.05% + 5%BSA. Nothing seems work.
I incubate with anti-LC3 Ab O/N at 4C (or 1 hr at RT); wash 3 times for 5 min with TBST 0.05%; add secondary HRP conjugated for 1.5 hr at RT; wash 3 times for 5 min with TBST 0.05%.
Add ECL solution for 5 min RT and use a chemi-doc for visualizing.

Please I really appreciate any suggestion...I'm like walking in the dark!No more ideas... :unsure:




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