I am trying to optimise concentration of chloroquine ()to assess inhibition of degradation of my protein) in cycloheximide chase assay for HeLa only and HeLa -B*38/39-HA. Unfortunately, i can't get any signal for the antibody. Looking forward for help.
Regards.
LC3 is involved in formation of autophagosomal vacuoles (autophagosomes) and therefore used to monitor the formation of autophagosomes. The LC3-B is membrane bound, hence to determine it, its very important that the membrane fraction is not lost during sampling for WB.
Hope, you might have followed the specifications given by the company for anti-LC3-B antibody to locate LC3-B on WB (about 15 kDa), while the loading control of GAPDH (about 36 kDa) is clearly seen in your WB image.
The following issues may be fixed for the purpose:
1) Check whether chloroquine issue is not the factor for your concern
2) Check whether you have used 0.2 µ PVDF membrane (larger pore size of membrane may allow the 15 kDa LC3-B to pass out from membrane while transfer
3) Did you get band corresponding to 15 kDa after protein transfer on the membrane with ponceau stain
4) If not, increase the total protein per lane as well as transfer time and also check the transfer buffer having sufficient ions
5) If still you have problem, then fix the antibody issue by replacing it with the fresh anti-LC3-B antibody in order to rule out the antibody issue.
6) Recheck the source and concentration of secondary antibody also.
Good luck
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