There is no trick to this. As long as you use a reasonably high % acrylamide gel (12% will be fine) you should be ok. You do need to make consistently good quality gels (you could try pre-cast) and get the loading right - if the proteins do not resolve well at the low Mw range, you will struggle to resolve the LC3 bands. We have tried making cell lysates (RIPA buffer) and direct lysing into SDS-PAGE sample buffer, with no noticeable differences. I would recommend not freeze/thawing protein samples, and be aware that some antibodies recognise LC3-II with greater affinity than they do LC3-I. It has become popular to measure LC3-II normalised against tubulin or actin, but ensure that you do not quantitate samples across multiple gels. You may like to consult the Autophagy Forum at the Autophagy journal (https://www.landesbioscience.com/journals/autophagy/forum/) as this has info on some commercial LC3 antibodies.

It is also possible that the antibody you are using is not the best possible one. Have you tried any other antibodies against LC3? You can check autophagy forum for tips on antibodies:

http://www.landesbioscience.com/journals/autophagy/forum/?nocache=1887078014

I used: a) pre-cast gel (4-15%) from Bio-Rad; b) a LC3 antibody from Novus.

Baharia is right. As shown by the blots, treatment with CQ or Baf-A1 also increases LC3-I levels compared with UT, in addition to increased conversion of LC3-I into II. Thus, the way I used might not detect all LC3-I in untreated cells.

I used Ripa buffer containing protease inhibitor, and used high acrylamide gel (above 12.5% gel). Be aware of using small pore size of membrane since the m.w. of LC3-II is too small, thus pore size of membrane is critical for detection of LC3-II. Both LC3-II antibody from Novus and Cell signaling is good!

Plus, I recommend you to measure SQSTM1/p62 for evaluation of autophagic flux. As you know, p62 is known as autophagy substrate, maybe the easy way of observing autophagic flux!

I agree with Baharia, lysate buffer is really important! As LC3-II is bounded to the membrane, do not ultra-centrifugate your sample.

And as Nari say, take care of other autophagy markers, like p62, mTor, beclin1...

Good luck with it.

Do you use PVDF to transfer? LC3-II is conjugated with phosphoethanolamine and become fat soluble. I have read PVDF will be better than nitrocellulose for LC3 western blot and I have used it ever since. Also, be aware of the pore size since the molecular weight will be 12-14Kd it can easily swim through the regular 0.45 um membrane. Voltage or current shuld be low for the same reason.

I used CellSignaling LC3B-XP which works well for my experiments. I run lysates on BioRad any-KD gel and transfer to PDVF membrane using 200A for 60 minutes (wet transfer). I am not promoting any specific product but like to share my experience. Serum starve or OPTI-MEM without serum can be used to induce autophagy. You can also block the completion of autophagy simply by adding NH4Cl (20-40 mcM).

Be aware, simply detecing P62/SQSTM1 may not be easy to interpret if you fail to manipulate the autophgic stress. There are some newly developed indicators (Enzo) you can use and quantify autophagy by microscope. But, I have to admit that I have not yet try those new methods yet. Beclin-1 also modulate apoptosis so to interpret the result may also be not easy.

Thanks for all answers. Ru-jeng Teng, I have wonder about NH4Cl how to use it(Final conc., treated time after we induce autophagy.

I don't know how much LC3 express in HL-60. And I guess that it's completed autophagic process so why I didn't see any thing.

Today I will try it again,TT

thanks again for all comments

The NH4Cl concentration (20 mcM final) I used in my publication was from Chen Y et al. J Cell Sci 2007;120:4155-4166 and treated for 2-4h. I will be hard to predict how much LC3 will change by my own experience so I always have more than 2 groups to compare. Maybe you have to serum deprive, even take out the glucose, for 2 hours to see what will happen. I have never work on HL-60 so can not give you any suggestion regarding it. Did you look into any publication studying HL-60 autophagy? I just goggle "autophagy and HL-60" and found several recent publications available. Feel free to do so and find out their experience.

I think that is too importante the buffer lysis you use to detect autophagy markers, we have some problems depends on the samples. You should know the autophagy turnover in your model, so that's a good option use inducer o inhibitor, to observe the conversion LC3-I in LC3-II. RIPA búffer anda SDS al 12% works ok, and sigma ab.

I use wet tank transfer method under 200-225 mA for 60 minutes to PVDF membrane. I found this gives me the strongest signals. The antibody I used was LC3-XP from Cell Signaling just because my collaborators used it. Using this transfer I can also get signals for acetylated-tubulin and SQSTM-1/P62. RIPA maybe a better lysis buffer since it is one of the strongest lysis buffer but I have changed to MOPS buffer which also gives me good signals.

dear guys,

i also have a problem with detecting the LC3II band on my western blot.

the lC3I is not well separated from my LC3II band or the LC3II band is a smier!

I use 4-20%Tris/Glycine gels from Biorad.For the lysis step we use MPER extraction buffer with proteinase and phosphatase inhibitors. I use 75Volt for the SDS-running, 35Volt for the transfer time.I also use PVDF membranes.But I do not know if I should chnage the running,transfer buffer or the lysis buffer for the sample preparation.

Thank you for your help!

Did you pre-wet the PVDF with methanol? I use regular running/transfer buffer from BioRad without any problem. Personally never had experience with MPER buffer and can not comment on that. I also use TGX but run at 200V for 45 minutes which works fine to me. I transfer by 250 mA for 60 minutes using wet-tank method.

yes i pre-wet my PVDF membrane for 3min with methanol.yes i am also using wet-blot for 1,40 min. Which regular running/transfer buffers from Biorad are you using exactly?? did you mean ( 10x Tris/Glycine /161-0734/ premixed electrophoresis buffer and also a ready to use transfer buffer)

We had self prepared running and transfer buffers in our lab.i can look it up tomorrow you many we exactly weight in from each substance.

We use 10X Tris/Glycine/SDS buffer cat#161-0732. I have never used the 161-0734 and can not comment on whether that will make the difference or not. But they do have difference in SDS. Judging from LC3B-II is PE-conjugated LC3B-1 and very lipophilic the SDS might make a difference.

The 161-0734 must be for transfer with 20% methanol in it.

hi we are using for 10x Running Buffer these ingredients: 144g Glycine,

30g TrisBase,10g SDS pH= 8.3 (with 6M HCL)--> and then we dilute this to 1x runing buffer.The transfer buffer is: 30g TrisBase, 144g Glycine pH=8.3 (with 6M HCL) --> then we dilute this to 1x transfer buffer.

But I think i will order this 10X Tris/Glycine/SDS buffer cat#161-0732 and try it!!

And for Transfer Buffer which LOT numer are you using??

thank you!!!

#161-0734 is the one we used for transfer. I could not find the LOT number on the bottle. The control #200006709 and 200006780 are the numbers I could find on the bottles.

LC-3 protein is found in membrane and aqeous conditions depending how active your lysosomal flux. You need a lysis buffer which efficiently extracts membrane and cytosolic proteins. RIPA buffer an be used with good confidence to excract proteins like these. I am not very positive about MPER Buffer.

Thank you all for your answers.

Also, You may improve your transfer efficiency if you add 0.01-0.03% SDS to your transfer buffer (for semi dry transfer). I have not optimized this concentration for wet transfer. You may try similar concentrations of SDS. Efficient transfer is very important when you are trying to transfer membrane bound proteins, in this case LC3. Good luck with your experiments.

Hi, my name is Elena and I also have a problem with WB LC3.

I obtain two clearly visible and nice bands, but they results to have a 10 fold higher molecular weight ( about 100-120 kDa).

I use the following buffer for lysis: 150mM NaCl, 50 mM TrisHCl pH 7.4, 0.1% SDS, 1% triton x100, 1% Na deoxicholate.

Do you think is ok?

Thank you very much for your help.....

Elena

Elena, I am having a similar problem, though my main bands show up around 50 and 60 kDa. I do have bands also at 25 and 30 kDa. I have looked for mentions of larger molecular weight forms of LC3 or some other explanation for this, but have not yet found anything.

Katherine, I've read something about hydrophobicity of LC3, but I cannot download refs, tomorrow (when I'll be in lab) I'll send to you the references, maybe you can download them, wich lysis buffer do you use

Hi Katherine,

cited from ‘Guidelines for the use and interpretation of assays for monitoring autophagy’ Autophagy 8:4, 445–544; April 2012:

‘ Caveats regarding detection of LC3 by western blotting have been covered in a review.

For example, PVDF membranes may result in a stronger LC3-II retention than nitrocellulose membranes, possibly due to a higher affinity for hydrophobic proteins (Fig. 5B; Kovsan J, Rudich A, personal communication), and Triton X-100 may not efficiently solubilize LC3-II in some systems.[141] Heating in the presence of 1% SDS, or analysis of membrane fractions,[142] may assist in the detection of this protein.’

I’m interested in reading ref no. 142: Tanida I, Ueno T, Kominami E. LC3 and Autophagy. Methods Mol Biol 2008; 445:77-88; PMID:18425443; http://dx.doi.org/10.1007/978-1-59745-157-4_4

I cannot download it.

I also wrote to the authors, but they didn’t answer…are you able to download this ref?

A antibody is coming which can detect both LC3-I/II. You will get two band, uper band is for LC3-I while lower represents LC3-II.

Hi pradip can you tell me the name of antibody which detects both lc3I and lc3II band on western blott...

its a company LC3A/B  from abcam. it is also known as LC3-I/II.

Catlogue ab58610-100

Thank you very much pradip i am really Grateful for Your quick reply...

You welcome Dr. Rizwan

Please use LC3 B specific monoclonal antibody for the detection.

Hi Everybody! We are also trying to standardize this WB. Could you please inform us how much protein do you charge on the gel?

You should load several concentrations of protein. You will see if you are inside the lineal part of the signal. I used to do 2.5, 5 and 10ug. 

Hi All

Awesome feedback on this tag so far and I would like to add a couple of following points:

1.    LC3-II form will only be detected in the samples under study if the cells are undergoing autophagic death. Lysates of chloroquine or NH4Cl treated cells or simply a total cell lysate from serum starved cells depicting excessive vacuolization are great options for use as positive control (shows both LC3-I & II bands).

2.    The major difference between LC3-I & II forms is their lipidation status, the conjugated PE moiety, and all LC3 antibodies are known to detect both forms. NB100-2220 with 430+ publications is the highest cited antibody in autophagy research field.

For more than 25 similar frequently asked questions on LC3/Autophagy analysis, visit Novus Support page “FAQs - Autophagy and LC3”: http://www.novusbio.com/support/faqs-autophagy-and-lc3  

Best Wishes

Subhash

I use 15% gel and LC3 antibody 1:1000, loading 10ug or  20ug of protein

I use 12% gel, and I load 40 ug of protein. LC3 antibody 1:1000

Nakatogawa, H., and Ohsumi, Y. (2012). SDS-PAGE techniques to study ubiquitin-like conjugation systems in yeast autophagy. Methods Mol Biol 832, 519-529.

Hi,

A lot of discussion on this already, I hope Patompong you have got your bands... I just came across this question and could relate to it..as I too had a lot of trouble with the LC3 bands...finally which I could solve with just two important points:

1. Using a 12% bis/acrylamide gel

2. Using PVDF with 0.2micro m pore size.

3. I used 50micro gm of protein samples.

This should help you !

I want to detect LC3 B proteins from the HEK293 cell line. So, how to extract LC3 A/B proteins from HEK293 cell line and which antibody should I use for western Blot ?

Is RIPA buffer only okay for WB or do I need to do sonication after that as well??

thanks!

Shahid Shahid Better you go for sonication as well in addition to RIPA Buffer. Increases the yield.

Subhash Gangar Hi 👋

Is Hydroxychloroquine ok to use as positive control of LC3 detection?if yes ,which concentration?I am working on nalm6 cell line