Hi,
I have recently encountered a loading problem with one of my blots. The data just didnt make any sense. I reprobed for actin. The loading problem should be correctable for actin normalization but it is not fixing the problem. I attached the image of actin. Yes, it looks bad.
I ve done this many times before and it works beautifully. Had many probelms with other things, but not this one problem. The protocol was the same thoughtout. The only difference this time, was that I changed the stripping conditions from more harsh (using b-me buffer) to milder (using 0.1M glycine at pH2.5)
I am very careful when loading my samples, and the samples are normalized to load the same volume/ amount for each well
I am perplexed to what could be causing this problem.
Could it be the transfer issue?
I havent altered my transfer conditions though. 60min 90V
I dont stain the membrane, but I do stain the gel. It shows no bands left in the
range of the molecular weight I work. There are some bands at the very top, but my protein is much lighter.
Please let me know if you have any thoughts.
Many thanks
Looking at your question I am assuming that the protein for which you are initially probing, before probing for actin, looks fine on the pre-stripped blot image.
You say that the protein is "lighter"; does it run around the same place as the actin? If so, perhaps the problem is that your less-stringent stripping conditions are not suitably removing the antibodies that bind it, thus impacting the way the actin band looks. My suggestion would be to do two blots, probe and image the original protein, then do a strip with both the original, more stringent conditions on one blot, and a strip with the less stringent conditions on the other blot, and then expose the blots to your ECL or similar reagent, get an image for the two blots and see if you did indeed remove the antibodies successfully.
Since you have mentioned that the only change you made was in the stripping conditions, that would seem to be the most likely source of the problem. Otherwise, perhaps one of the reagents you are using in the blotting process needs to be replaced/re-made. Also, make sure (if you are not already) that you clean out the wells of the gels prior to loading sample by pipetting/syringing buffer into the wells; if your original protein looks fine, as assumed above, that is unlikely to be the problem but it is always a good procedure.
Hi, Aleks, what do you mean as "The data just didn't make any sense"? How was looked the first scan (before striping)? If in the first picture bands were sharp, it mean that something wrong with the striping/re-probing. alternatively, you have a problem with loading on the gel.
Please, wash out membrane in water and stain it with PONCEAU. You will immediately answer all your questions about loading, transfer and washing procedure! You can try for second staining NBT-BcIP method. I this case you will not have interference with the first staining. Good luck!
Dear Aleks,
Your bands are perfect. Your "The data just didnt make any sense." I would change into: "It did'n't match my expectations". Could be the actin in your experiment is regulated.... Gapdh or ponceau will resolve this problem.
Best - jan
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