Question

Problem in Ponceau staining after protein Transfer

Very recently I noticed that even if I have enough protein on the gel, enough to be detected by commassie stain- I do not get it stained by Ponceau on the blot after transfer. The protein gets transferred completely and the bands are visible after the western. I also ensured that the protein does not get lost due to over transfer. Western is working quite fine, initial protein concentration is good enough up to 50ug total protein, total transfer of protein does take place, there is no heating up during transfer, pH of the buffers are fine - I do not understand what might be the problem.

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  • Kalpita Karan · Jawaharlal Nehru Centre for Advanced Scientific Research
    Hi,

    I faced the similar issue but even I am still not sure what the problem is. What I usually see is that when there is a problem in current input as in the current -voltage is maintained fine but its low over time, it happens. What I mean is when I transfer my protein which is 70kDa, for say 1 hour, the current drops from initially what it was to very low and when I changed my transfer apparatus to another, it seems fine now.

    This is just my observation. You could probably try in another lab or apparatus and see if its a power issue. Proteins will be transferred but then the rate might matter.
  • Varsha Mohanan · The Maharaja Sayajirao University of Baroda
    Thankyou Kalpita for sharing your experience.
    But I don't have such a problem with input current, moreover I do not think there might be a problem with the apparatus because our apparatus is quite new. Transfer is fine, no heating up...!!
  • Don Beezhold · Centers for Disease Control and Prevention
    Had the same issue with a fungal proteins, we concluded that the protein was transferring (positive Western) but did not bind Ponceau dye for some reason.
  • Antonio Ruiz · University of Alcalá
    Are you transferring to nitrocellulose or to PVDF membranes? Proteins in PVDF membranes are not well stained with ponceou.
  • Carlo Martins · University of São Paulo
    Is your Ponceau made "in house" or do you buy a commercial reagent? Maybe is the pH... I had the same problem, with "in house" reagent: I made another batch and it started to work fine! I hope this can help you!
  • Darien García Díaz · Center for Genetic Engineering and Biotechnology
    Hi, could you tell us some deteails of your experiment? Transfer buffer composition, ponceau solution composition, type of membrane....? For example, if you are use PVDF membranes like Antonio Ruiz said, you will need to ativated your membrane after transfer, immersing in methanol before a wash with water or PBS and the ponceau staining. This not happens with nitrocelulose membranes...also some reserachers have reported some interferences with ponceau staining if you use SDS in transfer buffer....
  • Giuseppe Di Maio · National Research Council
    I suppose that you're usig semi-dry blotting apparatus. Try to use another similar apparatus or wet-blot (It could be the power supply or something else). Change the ponceou solution or ask about a ready made one (if you're using one made by yourself). I can't see many other points in between that you've not already checked (or have been already suggested). Is it the same with some other sample?
  • Nikhil KL · Jawaharlal Nehru Centre for Advanced Scientific Research
    Like most of the above answers, I think there must be some issue with the ponceau solution itself or probably the membrane you are using is not suitable for ponceau. I mean in case you are using nylon memb, then that answers your issue. Given that you say your western seems to be fine, It probably has nothing to do with the apparatus or transfer buffer (again because you said western is working fine). Or recheck your ponceau, try a new set of solution to check if your solution is contaminated. Any contamination with positively charged molecules is sufficient to screw up the solution. Try a different protein as well..good luck!!
  • Julius Dongdem · University of Nottingham
    Is your ponceau S = 0.1% (w/v) in 5.0% acetic acid? And do you immerse membrane up to 1 hr with shaking. Definately, PVDF created problems for me too.
  • Varsha Mohanan · The Maharaja Sayajirao University of Baroda
    Thanks everybody, for suggestions. Darien your suggestion did the job. its the PVDF membrane. I have been using PVDF membrane for western since last 4 years, but this problem never occurred to me. The new lot seemed to create problems. Thanks Darien!!
  • Darien García Díaz · Center for Genetic Engineering and Biotechnology
    Anytime!!
  • Juhura Almazi · University of Sydney
    In Addition to all previous suggestions, I have SDS in my transfer buffer and use PVDF membrane for my western blots and I find that using the Sypro Ruby protein blot stain is much better, however you will need to have a scanner that can detect 'SYPRO RUBY' to visualise it.
  • Nicolas Casadei · University of Tuebingen
    Personally I prefer the amido black staining, much more sensitive that the ponceau!

    http://www.sciencegateway.org/protocols/cellbio/protein/sp.htm


    http://books.google.de/books?id=nI1RSmX0GXUC&pg=PA73&lpg=PA73&dq=amido+black+ponceau+silver+staining&source=bl&ots=sbf5KO89Lx&sig=3Y9DmEI5OyenbbIG7ec0A6vKXwQ&hl=en&sa=X&ei=EY6TUN67AcTmtQbvjIGgBA&ved=0CDUQ6AEwAw#v=onepage&q=amido%20black%20ponceau%20silver%20staining&f=false

    Best regards

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