Hello protein experts,
I use a 3-14% gradient SDS-PAGE gel to resolve my samples. The gel usually works fine. However, recently, I'm having trouble detecting proteins in a bigger size. The bands below 50kDa are usually fine but not the ones above. I use these gels in western blot. Before transfer, I cannot see the marker bands in the top haf of the gel. After transfer onto a nitrocellulose membrane, I can barely see any bands on the top half of the membrane with Ponceau stain or antibodies probing. I have made new buffers for the gel but the problem continues. I hope someone can give me an idea on what's happening with my gels. Thanks.
Hi,
I think the transfer from gel to the membrane is not sufficient, can you say what type of transfer you are doing, the system, power and how long. Is your ladder is transferred completely including the top bands.
If yes and only proteins from your samples are not transfered then you need to consider this, certain protein likes methanol and SDS in the transfer buffer certain does not. In that case do a commassive of your gel after transfer then you will know about the top bands does it stayed back in the gel or not.
A little bit research will help you to resolve the problem. Good luck
Hi Xiaojun Yuan
I agree with Hakim Jafferali. If insufficient transfer issue is there then you can try overnight transfer protocol which easily transfers the high molecular weight proteins. However you must also check the integrity of your sample. as high molecular weight proteins are more prone to degradation.
I'll disagree with the above answers. If you write you do not see bands on the gel before transfer, then the transfer is not to be blamed.
This seems like problem with your SDS-PAGE. If I understand correctly, you have run these gradient gels previously and they worked fine, right?
Can you say, if the bands are only misplaced (is one of them of different color to be identified?) or missing altogether? What does it do during run? What if you turned it off earlier, let's say in half of the usual time?
Thank you guys so much for your reply.
I dont think it's the transfer since I cannot even see the marker bands before transfer. And I coomassie stained the gels before transfer today and I cannot see my samples either. So it's the gel.
I have been running these gels for a year and they have been working perfectly fine for me. We use duel-color pre-stained markers so it'e very easy to identify them. Since we run these gels overnight (~17hrs), I'm not sure what happened to them during running. Sometimes if I come in early and gels still need another 4 hrs, I still cannot see the marker bands.
What kind of gels is that, that you run them overnight? Just curious.
Anyway, I suppose you have fresh gels, right? Are those bought or did you cast them by yourself? Could you contact the seller and tell them you have problems and you would request one or two gels from another batch(es)? That'd be ideal.
However, did you check the wells? Did you stain the whole gel including wells?
Can you post a picture of the stained gel? That could help.
Hi Tomáš
I cast these gels myself. These are large 20*18 cm gels. We prefer to run them slowly so they dont heat up that much.
I haven't check the wells. That's a good point. Although the samples on the bottom part of gels are perfect, I'm just wondering if it's possible for only the larger proteins to be stuck in the stacking.
I currentlt dont have an image for the stained gel.
Thanks for the reply.
Could it be possible that you would have switched the acrylamide solutions? I.e. to have high percentage at the top and low percentage in the bottom? But I suppose you'd see the proteins, even if at the top.
Hi Tomáš,
I dont think that's the case. Besides, it's happening to everyone in our lab. We're currently look at the power supplier. Not sure if that can mess up our gels.
If you plot distance traveled vs log molecular weight of the markers, starting with the 50 kD marker on down, is the slope the same as you had before the trouble? If not, chances are that the acrylamide or bis-acrylamide concentrations are not as you had it before. If everyone in the lab is casting their gels with the same solutions, then everyone would have the same problem. It would be easy to check by preparing new solutions, cast the gel, and run just pre-stained molecular weight markers.
In reference to my answer above, since you will be comparing different gels, it would be (distance from well to band / distance from well to solvent front) vs log mol wt of the marker represented by each band.
I think that I am having the same problem that you had on your question. Please, what did you do to solve that? What was the problem? Following a picture of my gel. Thanks!!!
I met the same problem these days, and I find out that there is some problem with my stacking gel .Larger marker separate in stacking gel and my protein looks ugly. May be you can change your stacking gel for a try
I have had this very same problem throughout my whole research life with a frequency of 10-15% of my gel runs. Nobody (including me!) could have ever come up with a clear answer! The problem is definitely about the run. The high molecular size pre-stained proteins (marker bands) pass to the resolving gel, but then they gradually disappear as run continues, as do the high molecular weight proteins in my sample. Many times I thought I figured out the reason, but none of those solutions have survived through time. Is it gel components? is it the water I use? Is it the sample loading buffer? Is it the running apparatus? I have tested many of such factors. Whenever I thought I am close to resolve this mystery, turned out I was far away from it...
I meet the same problems several times but not always, the gel I use 4-15% and Tris/Glycine/SDS buffer which all buy from Bio rad company. If I run long, I will lost more upper protein samples and marker. Hope some one can give me some suggestions! Thanks!
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