We are experiencing issues detecting LC3-I and LC3-II by WB. We have tried different antibodies (abcam and Novus) and we obtain the same results: very weird pattern in the bands we detect, particularly at the edges of the lanes (see attachment). We would appreciate any help in this.
Ruth
What % acrylamide is in the stacking gel? Making your stacking gel bigger might provide a better resolution.
Hi Mario,
Thank you for your quick reply. We have used 8%, 10% and 12% acrylamide. In all cases, we observe the same results
Hi Ruth,
You would certainly like to try Cell Signaling Abs. Please find the publication attached. Hope it will be of help. Feel free to ask further queries.
Article Quinacrine-mediated autophagy and apoptosis in colon cancer ...
Hi Ruth,
Uneven running. May be two things result in the bands. One is that how do you prepare the sample for WB. And another things is that the gel preparing.
Hi Shi,
We have used several methods to prepare the samples. (1) Lysis IP buffer (based on RIPA) from Thermo, (2) Lysis IP buffer supplemented with protease inhibitor cocktail and (3) Lysis IP buffer + proteases inhibitor cocktail +SDS + EDTA. In all instances we obtained the same results.
As I mentioned earlier, we have run the samples in 8%, 10% and 12% polyacrylamide. Again, no matter the acrylamide concentration we always obtain the same results.
Ruth, could u do the following?
5X SDS-PAGE buffer (10ml)
1M Tris-HCl (pH 6.8) = 0.6 ml,
50% Glycerol = 5 ml,
10% SDS = 2 ml,
Water = 0.9 ml,
2-mercaptoethanol = 0.5 ml,
1% bromophenol blue = 1 ml
Try making this. And make sure u're taking 80 microgram protein / sample. Good luck.
I think your sample is not dissolved when you load your sample. Is your target antigen sensitive to reduction? If not boil you sample for 5 minutes before you load it. Best to heat in glass tube. Alternatively, give the samples a spin at say 10,000 x g for a few minutes to bring down the ppt.
Good luck,
Butch
Hi Butch, thank you for the advice. We boil the samples before loading. We have no problems detecting other proteins with our technique. Only LC3. I will try spinning them down as well.
Ruth
Hi Ruth,
just a guess. It looks a little bit like a extreme form of dumbbell-shaped bands. The cause of this is often that the gel is too hot or the migration is too fast. So maybe, if you use less voltage or run the gel in the coldroom could improve your results. Since it is only the LC3, maybe pH is the source of the problem. If LC3 is "naturally" a highly charged protein could this, together with the charge of the LC3-bound SDS, lead to relatively fast migration compared to the "normal" protein. If in WB the apparent molecular weight of LC3 is lower than the calculated moleciular weight could this be a clue for this problem. Changing the pH a bit ( a bit lower) in the running buffer could be worth a try.
Good luck,
Carsten
Thank you, Carsten. This is a great suggestion. We will try decreasing the voltage and slightly changing the pH of the running buffer. I will keep you posted.
Ruth
We have finally used the cell signaling antibodies that most of you recommended and the results are amazing. We actually analyzed side by side the same samples with the antibodies we already had (Novus and Abcam) and the new ones from Cell Signaling. The resolution is fantastic. With the Novus antibody we barely see bands, and with he abcam antibody only one band is visible. The cell signaling antibodies allow us to easily distinguish LC3-I from LC3-II. I am so happy that we have finally found a good antibody to check autophagy by western blot. Thank you for your suggestions!
Ruth
- Badges
- Science method