I wanna detect two target proteins, one is 14KD and the other one is 15KD.I tried to use the 15% or 12% separating gel. But every time when I got the exposure imaging, the target band are crooked and adhesion (show in picture). Who have ever suffered these problem and worked out, please help me. Thank you very much. Waiting for your reply, sincerely.
Do you have a loading control such as GAPD, Beta Actin, Beta Tubulin, etc.? The loading control would help us understand whether the problem is unique to the antibody (doubtful), the region of the gel where the samples are resolved (possible, but doubtful), or an issue with sample preparation (more likely).
I think there's something off about your samples, but it's hard to tell what. It could be high salt, high loading, lipid contamination, etc. A well-behaved positive control might help get the assay working for your samples, if such a positive control is available.
Hi Riqiang Bao
Are you sure to look at the right height? It might be that you are looking at the front line of the protein sample. There are no separate protein bands visible. You do not indicate where the molecular weight markers did run in this particular gel/blot.
A 15% SDS-PAGE should be good enough to use for 14-15 kD proteins. Do not expect to see a difference between the height of both bands. Higher % might even lead to better separation of the proteins.
Are you sure the samples have the same buffer composition? Differences in salt concentration among SDS-PAGE samples often leads to ‘waving’ bands in the gel/blot. This might be the case for you too.
And now…
Indicate where in this image run the molweight markers. Are they pre-stained or non-stained?
Where are the positions of the slots
Is this the gel or a blot?
Good luck…
WOW. I guess the problem is from SDS-page.... you should check it
Hi Riqiang Bao
Are you sure to look at the right height? It might be that you are looking at the front line of the protein sample. There are no separate protein bands visible. You do not indicate where the molecular weight markers did run in this particular gel/blot.
A 15% SDS-PAGE should be good enough to use for 14-15 kD proteins. Do not expect to see a difference between the height of both bands. Higher % might even lead to better separation of the proteins.
Are you sure the samples have the same buffer composition? Differences in salt concentration among SDS-PAGE samples often leads to ‘waving’ bands in the gel/blot. This might be the case for you too.
And now…
Indicate where in this image run the molweight markers. Are they pre-stained or non-stained?
Where are the positions of the slots
Is this the gel or a blot?
Good luck…
Yup, I have got an beta-acting as a control.Like the picture i have uploaded. The beta-acting also aligned. So the antibody should be okay. At the same time I use the SDS-PAGE run the other sample (target protein are 30-60KD) the results are not bad.(Like the second picture showed.) So the gel should be okay. It's the blot, and i make sure at right hight. I have repeat experments three times and every time got totally same results.
Thank you Duco, Brain and Yi.
It looks like the front line of your running proteins. If you don't run long enough your small proteins will be still in this running edge. Marker would be very helpful here.
Well, I take it back. The beta actin does look okay. I'm inclined to agree with the others, that the weird appearance might simply be due to the location where the protein is running. One of my colleagues uses 4-20% gels to resolved LC3B, which is close to the mass you're looking for. Perhaps the gradient to 20% would be enough to resolve the product.
By using the marker, i am quite sure that I have run the separating gel long enough. Thank you Anna. And i will try to use the higher gradient gel or change the sample. In order to see weather it's helpful or not..Thanks for your help Brain.
According to my experience, even using 15% selfmade gels you should be able to separate 14 and 15 kDa, I am separating this LC3AB-I and -II with the molecular weight of 14 and 16 kDa regularily without any problem and there is sufficient space in between to get a 15 kDa band. A gradient gel might be even better but in principle a 15% gel should be fine.
Did you ever do some Ponceau or Comassie staining with your membrane after the blotting process as control for your blotting? Just to ensure how protein bands are looking on the low molecular weight? This is a simple but very nice control of your total protein and the single bands of it and also shows if you have air bubbles during the blotting. Up to the lowest molecular weight I always get very nice, sharp clean protein bands and when going for specific antibodies I also get very nice bands.
I regularly run 11kDa protein on a 15% gel and have clear separation from the dye front. We also use 10-20% gels. How long are you polymerizing your gel for? at least an hour, I hope, and at least 20 minutes for the stacking gel.
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