I perform dot blots by pipetting 1 uL of peptide solution onto nitrocellulose. I have one peptide that gives me a strange pattern - you see a faint dot in the middle, and also a larger ring around the dot (top right dot in the attached figure). No one I work with has seen this before. The only explanation I can think of is that maybe the protein doesn't bind well to the nitrocellulose, and it travels with the PBS as it moves through the membrane, basically depositing where there is no more solvent to carry it. It is important to note that all peptides in the attached figure have similar hydrophobicity, and sequence composition. I detect with a polyclonal primary and HRP-conjugated secondary. Any thoughts would be greatly appreciated.
I have experienced this before. It usually happened when I put too large of volume on the membrane as the capillary action pulls the peptide to the edges of the drop. I am not sure how you applied your sample, but I recommend repeated applications over the same area of very small volumes (0.5-1uL). Make sure it dries in between each application. This has helped me in the past reduce this phenomenon.
Good luck.
~Adam
I have experienced this before. It usually happened when I put too large of volume on the membrane as the capillary action pulls the peptide to the edges of the drop. I am not sure how you applied your sample, but I recommend repeated applications over the same area of very small volumes (0.5-1uL). Make sure it dries in between each application. This has helped me in the past reduce this phenomenon.
Good luck.
~Adam
Dear Gene
your peptide would certainly seem to have diffused and, as you suggest, has spread to the limit imposed by the diffusion of the PBS and then dried in place. I've never seen this before either, but I use PVDF (specifically Immobilon P from Millipore), rather than nitrocellulose.
The following paper has a good discussion on this phenomenon: Xiaoying Shen, et.al. Minimal Size of Coffee Ring Structure. J. Phys. Chem. B 2010, 114, 5269–5274. Alsu Cui used polymers to suppress this: Cui et.al. Suppression of the Coffee Ring Effect by Hydrosoluble Polymer Additives. ACS Appl. Mater. Interfaces 2012, 4, 2775−2780. Good luck.
Hi Gene
Yes - wet with methanol, then a good wash in distilled water. I usually prepare a batch of membranes and store them in H2O at room temperature. For blotting, I place them on a moistened filter paper, briefly place a dry filter paper over the top to remove surplus water, then apply droplets of protein.
Hello,
I am facing the same problem and my pattern looks exactly like the upper right of yours. There is 1% triton in my sample and I wonder it may do something. I would like to ask, is the problem solved?
Many thanks.
I had the same problem and now after using dot blot apparatus it is gone. I used 5-0 uL volume without apparatus by spotting sample onto membrane directly which use to give me ring like spots. Now with apparatus I use 100 uL of sample and I do not have rings but dot. Its perfect. The apparatus is from bio-rad.
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