It might have run out because it is lower in molecular weight. Higher gel percentage, shorter run times could help.

Hi,

I have been fighting with it for a long time. I used Tricine gradient gels (4%;10%;20%).

I do not recommend shorter running times because you might not obtain proper LC3II from LC3I separation. Run it slowly (no more than 80v) making sure that 10kDa marker band is just on the edge of the gel. It may take even ~12h for electrophoresis only.

I need to know what kind of samples you use. What is your detection system, antibodies, transfer method? Maybe than I would be able to give you more hints.

Good luck!

I use 12% bis/acrylamide 29:1 gels, transfer at 80V for 1.5hr. The antibody is also from Cell signaling. However I couldn't always reproduce it. Last time I ran 3 blots at the same time and only one of them got 2 separated bands of LC3I and LC3II, the other two which were run together in a same box only had one band. I guess the transfer condition is quite important. 

However, I can't see any bands without the presence of bafilomycin. I loaded 50 ug/25 ul which is a pretty decent amount of protein but I still can't get anything. Hopefully anyone can give me an advice how I should increase the signal intensity in my LC3II bands? 

I use fluorescent dyes with Licor Odyssey detection system and in order to improve band intensity I used signal enhancer (http://www.merckmillipore.com/PL/pl/product/SignalBoost™Immunoreaction-Enhancer-Kit,EMD_BIO-407207)

Not sure if you tried that but you may air-dry nitrocellulose membrane after transfer. Hydrophobic domains are than exposed allowing better Abs binding. 1h incubation in RT with primary Abs before 4C/over-night may also help.

I used PVDF 0.2 um membrane to get the most LC3 retention. So I guess I can't let the membrane dry out, can I? I may try your suggestion of the signal enhancer. Thanks! 

12% precasted gels at 160V works fine for me (Jurkat cells, Hela cells, HeK cells, liver and kidney samples). I use the nanotools anti LC3 antibody (we worked with abcam and cell signaling antibodies but the nanotools one is the better one). I also use the Licor Odyssey detection system, works very well. 

One tip: always use bafilomycin to block autolysosome formation! 

So last time I had me and an undergraduate in my lab run 4 gels at the same time to compare our results. We poured our own gels from the same recipe, loaded the same samples, ran the gels from the same power supply but different boxes and transferred the gels also from the same power supply but different boxes. The results were frustrating: She got double bands in LC3B, I got single bands in one blot and the other barely showed anything. For p62, she had bands, I didn't get anything. It seemed similar to the issue I got above. I guessed the trouble laid in the transfer box but I could see the ladder completely transferred to the membrane. What can be wrong with that? Do you need to check the transfer efficacy with Ponceau even when you already see the ladder clearly on the membrane? 

By the way, I saw some of you using nitrocellulose membrane. Do you use the 0.2 um one? Is that enough to keep the LC3B? I guess in this review they claimed that the results obtained from nitrocellulose vs PVDF membrane could be significantly different so they recommended using PVDF because of its higher hydrophobicity. But in this forum, some people say that the size of the membrane is enough. So I'm asking whether 0.2 um nitrocellulose is sufficient since to me, nitrocellulose membrane always give a much clearer blot. 

http://www.ncbi.nlm.nih.gov/pubmed/22966490

Hi Sergi, 

I usually prewet the membrane in MeOH 5 minutes before transfer. I didn't soak them in the transfer buffer before doing the transfer. The undergraduate did the same but she left the cassette sit in the transfer buffer for a bit longer so that we could run together. 

I just found this paragraph

"The choice is personal and both work very well. PVDF membranes require careful pre-treatment: cut the membrane to the appropriate size then soak it in methanol for 1–2 min. Incubate the membrane in ice-cold transfer buffer for 5 min. Failure to equilibrate the membrane in ice-cold transfer buffer will cause shrinking while transferring and a distorted pattern of transfer." 

So I think you got a point. Thank you for pointing out. I'll try to improve it next time. 

Thank you all for your suggestions. I found out that my proteins were not stable (probably due to less concentration of protease inhibitor I was using). I used 15% resolving gel; 1 hour transfer time (semi-dry transfer) onto PVDF membrane. However, I could only see LC3-I band while LC3-II was found to be in a complex form with one of my expected proteins. The bands were not as intense as I would expect, but then that's because I did not use Bafilomycin. I am sure adding Bafilomycin to the culture conditions would improve my result. Thanks once again for your input. :) 

Speaking of the protease inhibitor, how long can it last? According to the data sheet of Sigma, the reconstituted 10x can be stable for at least 2 weeks. But I don't know how long it can be reliable to use.