transfer of low molecular weight protein - (Nov/20/2013 )

Hi everybody,

we are just new to this forum! We are working to try to detect the LC3 band in our cell lysate ( we use vascular smooth muscle cells from rat). LC3 has two bands at 14 and 16 kDa. We can find a band in our positive control that we made just putting our cells on starving but we can just see a "ghost " band in our sample. We are using 15% Tris HCl gel running at 80V for 20 min and 100V for 90 min. We use 0.2 um PVDF and transfer for 2.5 hours at 25V 4°C. (We tried also transfer for 45 min at 100V 4°C but was even worse). Has anyone some suggestion ???? Thanks a lot

Kidney

-kidney-

Assuming that your positive control is detecting the LC3 protein that you are interested in, it would seem that the problem is not with the transfer, but rather with the amount of protein present in your samples.

How much protein do you load for the +ve and for the samples?

Are they lysed and treated in the same manner?

-bob1-

Some weeks ago I also did WB for LC3 detection (human sample) after starvation. I just follow our standard protocol for blotting.

EP: 10% gel (6% stacking), 10^6 cells, 20µl sample, 10min 80V, 80min 130V

Transfer: PVDF, 380mA (~100V) for 90min @ 4°C

The result was quite good.

-2xzwei-

So - degradation of the protein in the mean time?

-bob1-