Dear Ozge,

Comparing your blot with the western blots of cell signalling website, I think the second band from bottom corresponds to LC3b. You have got a different pattern, however, this seems normal to me since you have used different cell line.

Dear Özge Can ,

You will not get exact mol. wt of the protein in the western blotting. I do believe that your last two lanes below 22kda correspond to lc3b I/II. If you are getting lots of non-specific band like this, then plz always use one positive sample(plasmid or some drug treated sample) as well as one negative control in the beginning or end of the gel.

Regards

sarat

Hi Özge, indeed, the processed peptides are the two lower ones, but you are probably seeing also the precursor proteins, it looks like in these tumour cells autophagy is triumphant! LC3 processing is laborious, you have at least an original peptide of 27-28 kDa (one of the two bands above 22 kDa, possibly both, they seem to go together), but the band above 30 kDa is probably non specific, it is expressed at the same level in all samples, even when the LC3 bands are less (sample 3) or more (sample 4)... assuming you loaded equal amounts on the gel.

Cheers.

Hi,

Depending on size, it should be the last two lanes. ideally the molecular weights should be around 16 and 17kD..

The lane at (22kD) could be the unprocessed LC3..

I hope it helps!

Please use 15% SDS-PAGE gel for better separation, block the PVDF membrane with the blocking solution a little longer (as you are getting multiple bands). The bands should be 14 and 16 KD for LC3II and LCI respectively. Generally the antibody you have used is a good one and it does not give such high molecular weight nonspecific bands.

this is nice. sometime you will get non-specific bands. I think that last two bands are for LC3 processed bands. You can either increase the dilution of primary as well as secondary ab. Also was more times before detecting. You can also use less sensitive kit to develop the blot.