3 replies to this topic

[zky]

Hi guys,

I am doing LC3 western-blotting. Some, but not all, papers used E-64d and Pepstatin A containing HBSS to starve cells before collecting and lysing them. I am wondering if E-64d and Pepstatin A are required for LC3 western-blotting. Could these drugs cause anything artificial?

[dtimm]

Both E-64d and pepstatin A are protease inhibitors. This means that the autophagosomes you are looking for (LC3 is the membrane portion of the autophagosomes) will not be degraded by the lysosomes. This will give you an idea of the "flux" of autophagy (which I assume is what you're looking for). You can also find LC3 without these, but it will only give you a "snapshot" of what is going on, as opposed to having a quantitiation over time with flux.

[zky]

thank you very much, dtimm

[autocell]

dtimm, on May 6 2009, 08:23 PM, said:

Both E-64d and pepstatin A are protease inhibitors. This means that the autophagosomes you are looking for (LC3 is the membrane portion of the autophagosomes) will not be degraded by the lysosomes. This will give you an idea of the "flux" of autophagy (which I assume is what you're looking for). You can also find LC3 without these, but it will only give you a "snapshot" of what is going on, as opposed to having a quantitiation over time with flux.

Do you have idea on what concentration of AO is good for detecting autophagy in cells using FACS. In literature there is from 100 ng/ml to 100 mcg/ml, incubated for 15 min. Also, is it required to use compensation for detecting green and red signal from AOl? Because there is signal in all channels, from green to extreme red.

Is it possible to detect the autophagy proteins using FACS? Example: detecting LC3B, Beclin, ATG 5 or 12 or 7, using the primary antibodies (used for western blot), conjugated with fluoresence labelled secondary ab?


Thanks