Hello,
I am looking for some help with a Western Blot issue I have been experiencing. As seen in the photos, I often get these grainy/fuzzy/blurry bands, where they do not look like this in Coomassie stained gels of the proteins (bands are good there), and have not looked like that using Ponceau staining of the membrane after transfer (although I do not do the Ponceau staining every time, so I am not sure if the bands would ever appear like this using Ponceau). I have sometimes gotten sharp, good bands for these proteins, but many times get these type of bands. It has happened with fresh antibodies, different antibodies (e.g. monocloncal vs. polyclonal).
I am using semi-dry transfer (typically, 20V for 1 hr although I have tried some different transfer conditions to optimize for these small proteins I am working with) with buffer containing 25 mM Tris, 192 mM glycine, 20% methanol onto 0.2 uM PVDF membrane. I usually block in 3% BSA and have primary and secondary Ab diluted in 3% BSA (have tried just a few times blocking in 5% milk, 2nd Ab in 5% milk with ECL detection).
I am not sure if this is some type of transfer issue or Ab issue or something else, though as I said the bands in the membrane seem ok after transfer when I have done Ponceau, and this issue happens with different and freshly prepared Ab. It seems almost random that I occasionally get good bands versus getting bands like this, so whatever the issue is I am unsure why it doesn't occur every time. These are smaller proteins I am working with (~5 kD and ~10 kD), so I am not sure if there is something due to this, as larger proteins I have worked with and other larger proteins others in my lab have done Western Blot for seem to never have this type of issue.
Thank you for any advice or suggestions!
Jessica Lipponen please check previous answers to a similar question (https://www.researchgate.net/post/Western-blot-speckled-bands) Since you mentioned new and old Abs give you this variably it might be Ab aggregates. You haven't mentioned what proteins you are looking for (may cross react with blocker) or whether this occurs after stripping membrane (can cause gaps in bands but not fuzziness). Hope thus helps.
Something might go wrong with you gel or membrane or the transfer system, as you can see the marker bands are not clear. I have never seen marker bands like this.
Besides, as you are working with 5-10 kDa protein, I would recommend using 15% PAGE gel and do not run them to the buttom.
Good luck!
for small proteins the use of tris-tricine gels will give you a better resolution and transfert on Pvdf membrane in caps buffer (10mM pH11) +10% ethanol is better for small protein too
Thank you all for your suggestions.
I have spun down the antibodies so I am hoping aggregates are not the problem. I have not done any stripping of the membrane. These are his-tagged and flag-tagged PTH variants we are looking at.
I am using 16.5% pre-cast Tris/Tricine gels, which have definitely given the best band especially for the 5 kD protein by staining, and run them according the recommendations of BioRad who makes the gels. I definitely get good bands on these gels by Coomassie staining of the gels, and when I have done Ponceau staining, the bands look good as well. I think the markers in the second two photos are relatively clear or at least how they normally look for me. I know I have seen the CAPS buffer suggested as well, so I could try that, but given that Ponceau staining of the membrane after transfer has given good bands, I feel like efficiency of transfer onto the membrane is at least not the main issue here.
I am still trying to think if I can identify any other issue.
Thank you all for your suggestions and advice!
I recently tried transferring onto 0.45 uM nitrocellulose membrane (I was using 0.2 uM PVDF), and this seemed to work much better. I am going to repeat/try again to confirm this is definitely the answer to this problem, but I got good, clear bands on the nitrocellulose.
Katja, thanks for your suggestions. I have tried staining with Ponceau after finishing the Western Blot and haven't seen anything (but this was on PVDF, which it seems these proteins may have been washed out from), so maybe I will try with nitrocellulose to see if the bands are retained well still after finishing the Western Blot. The gels always look fine, not melted, after transfer - and I did try shorter transfer times, which yielded some protein on the membrane as seen by Ponceau, but we found that the loner transfer time definitely gives more signal/protein on the membrane.
Thanks again, everyone, and hopefully the nitrocellulose membrane continues to yield good results!
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