I'm trying to do western blot for LC3B protein after the treatment of the specific chemical. In my knowledge, LC3b is a cytosolic soluble protein and become a part of autophagosome membrane in the autophagy process. Then, if I performed the subcellular fractionation(differential centrifugation) during cell lysis process, is it enough to use 'membrane-related fraction' regarding the autophagy activity? Or, should I use the both 'cytosolic fraction' and 'membrane-related fraction'? ^^;
Thank you. ^^ ~
Hello Jun-Soon Kim
LC3B gets lipidated into the autophagosomal membrane upon initiation of autophagy. I would just use the use whole cell lysate at first to see, if the Western blotting protocol with your current antibody, sample preparation and buffer usage works. Another problem with sub cellular fractionation is that you may lose vesicular structures, unless the centrifugation is optimized to get what fraction you are most interested in. There are some LC3 antibodies that detect both soluble and lipidated fractions in WB, wherein the LC3B (lipidated) runs below the cytosolic LC3 band. There are other monoclonal antibodies that specifically detect lipidated LC3, in case you would like to compare both populations of LC3 in your sample.
Hope this helps
Aparajita
Hi,
use RIPA buffer with 1% SDS to prepare whole cell lysate.
Be careful to separate proteins well on SDS-PAGE to be able to see both LC3B forms. Consider using 0.2 um nitrocellulose or PVDF membrane.
It might be beneficial to use lysate from ATG5 KO cells as control, as some LC3B antibodies are not that good.
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