I've been blotting for LC3B repeatedly and I sometimes get this problem where I have a blurred band around the LC3II mark and no sign of LC3I. Now that could be the two bands blurring together or it could be just that there's too much signal for LC3II for LC3I to show. I notice when running my gel that the ladder is starting to blur from the 35kDa point onwards. I'm using a 15% gel and tris-glycine buffer. I have tried running my gels slowly (at 70V) and at a normal pace (120V). I don't think the problem is the transfer or samples as the blurring is showing in my ladder and I have had this work previously with distinct bands for LC3I and II before. Can anyone please give me some explanation as to what is going on here because I'm driving myself crazy trying to figure this out. As you can see from the attached picture, my B-Actin (top) is a normal clear band, but the LC3 (bottom) is just a mess.
Dear Aisling
Have you thought about running a gradient acrylamide gel: say 4% To 20%. That might give you sharper bands for both disparate sizes
If the blurring is also present in the ladder, then some of these things may occur:
-the gel is not OK. Try different % (file as attacment!) .
-the buffer is not OK. Here is a recipie that surely works:
10X running buffer (“10X SDS buffer” or Tris/Glycine/SDS): 1L
30.3 g Tris (= 25 mM Tris in 1x running buffer)
144 g glycine (= 192 mM glycine in 1x running buffer)
10 g SDS (= 0.1% SDS in 1x running buffer)
o The pH of the buffer should be 8.3 if carefully prepared so no pH adjustment is required.
o Store the running buffer at room temperature.
Dilute to 1X before use!
-lastly... this looks like protein degradation. Do you work always on ice? Have you checked if the temperature of the buffer goes really high when you run the gel? that can cause protein degradation and smears
Your b.actin looks very nice, good job.
I like how you cut your pvdf, genus. I also do this.
Usually your primary will be in excess, i would recycle until the ones with milk curdled. But if you recycle, make a fresh one.
Maybe protein degradation, i would use inhibitors working cold , but after protein assay, to total equil for all samples, i add western sample buffer and boil to prevent decay, bexause it permently denatures here.
Some times the data is the data... could.be a phosphorlated/dephospho form of your protein. I am not familiar with this one, but erk would be an example..
Best Wishes!
Also.increase time for primary antibody incubation, like overnight at 4c
Your gel looks more than ok. A gradient would help. Are you sure that the problem is not in the sample (degradation, differential glycosylation, etc.)?
Thanks for all the helpful suggestions. I've made entirely new buffers (I am pouring my own gels yes) and the results are slightly better! The frustration for me is that I have been consistently running 15% gels and I have had several great blots with this method showing the two distinct LC3B bands. I am now seeing the two bands but they're curved, 'smiling' bands so i think it was likely a mix of gone off buffers and over loading protein. I had been loading 20ug of protein but I may lower this to 10 or 15. Aside from all this there may be some protein degradation in my samples as my RIPA buffer may have gone off too but I'll know once I've made new samples in fresh RIPA. I'll update everyone once I've done this incase anyone has had the same problem. I've noticed a lot of similar problems with low MW proteins in other forums.
Decreasing your sample load would be a good idea, especially if you pick up a good detection system.
For the “smiling bands“ , also try to manipulate the voltage and the running time.
I forgot to update everyone on how this got resolved. It was a combination of two things.
1. I had been using a 10x running buffer containing SDS and changed to only adding SDS into my 1x buffer. It seems that over time the 10x solution had been going slightly off and resulted in far too much salt in my 1x solution.
2. My Laemmli buffer was self made and may have been a bad batch. I've since bought in some premade Laemmli buffer which works wonderfully. (Bio-rad; #1610747)
I'm not sure if this will help other people in the future but this fixed my problem.
I too repeatedly ran in to this problem with low molecular weight proteins on my poured gel. Gradient gels from Bio-rad helped.
Hi Charles, I ran in to this problem when dealing with cleaved caspase 8 (M.Wt. 10) and other low abundant proteins below 25 kDa. I was able to address the problem by running a 15% gel to 3/4th of the gel and transfer them using 20% methanol containing transfer buffer for 30 mins with transfer conditions 1V/cm2 of transfer membrane. A matter of caution is, use this blot only to probe low molecular weight proteins as many of the high molecular weight proteins (35+) will not transfer efficiently. I hope it helps.
Small MW proteins can intercalate with free DS (from SDS) ions and get "fuzzy." I would suggest Tricine gels or gradient gels (e.g., 4-20%).
Also, this is unlikely but maybe you're transferring for too long?
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