Before running the SDS-PAGE, I added pre-stained Kaleidoscope on the well corresponding to the ladder, 30ug of proteins from my cell lysate samples and then as another marker, I added again pre-stained kaleodoscope on the last well after my sample (to make it easier for me to cut the membrane eventually). Both kaleidoscope ladders transferred successfully onto the membrane. However, I stained my PVDF membrane with Ponceau S for around 20 minutes and nothing could be seen relating to the bands.
If the ladder transferred, then most likely the proteins transferred as well even if Ponceau S could not detect it?
Was 20 minutes of Poncea S too little time?
Generally 20 mins of Poncaeu S staining should be sufficient to observe protein bands. Important things to make note of is
1. Was the Ponceau stain freshly prepared? It is important that the stain is as fresh as possible to ensure proper staining.
2. You could try staining for longer, say 30 mins in shaking conditions.
I would suggest you to nonetheless proceed with the western blot and see whether your antibody detects your band of interest; your loading control antibody (actin, GAPDH, tubulin etc) would easily allow you to judge whether there was any issue with the transfer.
Good luck with your experiments.
Sure, if enough proteins are in your sample they should be also transfered to the membrane.
However, I also see some possible problems:
a) your staining solution does possibly not work (maybe due to contamination, wrong formulation, maybe was not freshly prepared or other possible error sources.
b) Have you measured and verified presence and concentration of proteins in your sample?
c) other mistakes within the experimental or technical setting (apparatures do not correctly work, wrong kind of membrane (maybe PVDF vs nitrocellulose etc.), the experimentator itself and so on...
Thus, you can further perform WB and see whats happen (this will give you information whether protein was in the sample or maybe not, whether the staining was incorrectly done or possibly a the source of error etc.
Or you stop at this point and rerun the experiment and see whether the next try works better.
The first thing I'd do is to rerun the electrophoresis and stain the gel, to verify the presence of protein. You can use reversible staining for example with Zn/imidazole (doi:10.1016/0014-5793(92)80309-5), then you can use the same gel for later blotting.
You can also use india ink to stain the blot (doi:10.1016/0003-2697(83)90237-3). This is more sensitive than Ponceau, but is permanent (you can't use antibodies on that blot).
Ponceau solutions need not be fresh, a 0.5% solution in 1% acetic acid (doi:10.1016/j.ab.2010.02.036) is almost indefinitely stable at RT when kept well stoppered.
Hi
30ug of proteins are sufficient to be seen in WB, however in your case you have to know the size of your protein, if its too small and you run it for long time or high V you could be lost your protein. usually I run my sample overnight with 25V and I can see all my proteins large to small
another thing if your protein is small less than 25kDa try to cast gel 12% not 10% acrylamide
Hi, Eduardo Alvarez.
One question only. Although you have used kaleidoscope molecular marker and it's collored, when you stained with pounceau S, your kaleidoscope bands were collored by Ponceau S??? If not, Ponceau S was the problem and keep on going with your WB.
All other tests recommended are important to resolve your Ponceau S staining problem but If your kaleidoscope was visible (all bands from beginning to the end of the gel), you probably had problems just with the staining. Ok?
All the best.
Adriano Costa de Alcantara After using Ponceau S, the kaleidoscope bands remained the same. You can see each individual color of the kaleidoscope while the membrane is stained with Ponceau S.
Thank you all, maybe the problem is a combination of the Poncea S + blocking buffer solution. Maybe I have to rinse the membrane more since particles of milk can be seen attached to the membrane.
You do use the Ponceau before blocking with milk, don't you? Otherwise, the bound milk protein will cause so much background staining that you can't see weaker bands.
There should not be any particles in the blocking buffer, if necessary, filter it before use.
Hi, Eduardo.
Yes, as Engelbert said, you shouldn't block and then go to Ponceau S. It should be the other way around. Ok?
I do consider everything has gone alright with your transfer If you can still see your kaleidoscope marker but can you send us a pic of your gel and WB for evaluation of other details?
All the best.
Do you used stained molecular marker? This marker can be a reference to transfer. And you can stain de gel with coomassie to check the transfer
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