Hello,
I am encountering a strange horizontal smear of neighboring wells of my recent westerns. Now protein quantification is problematic. I am unsure whether this is due to some horizontal "leaking" of the wells or it is due to crowding of the primary antibody. It is unlikely that the problem is with the SDS gel since there are no apparent horizontal smears on the ponceau stained membrane albeit it is much less sensitive. Has anyone encountered this scenario? I am going to reduce the primary antibody concentration; however, I have been using the same antibody concentration before on numerous occasions and did not see this smearing. I appreciate your input.
Cheers,
Ladan
The ghosting artefacts associated with overabundance of proteins of interest in Western blots can skew findings. Since ghosting is associated with overabundance of the target protein, it should be addressed by reducing the amount of lysate that is loaded into SDS-PAGE gels.
For more detail please refer to published paper by Butler et al., 2019:
Misleading Westerns: Common Quantification Mistakes in Western Blot Densitometry and Proposed Corrective Measures. BioMed Res. Intl; Volume 2019 |Article ID 5214821 | https://doi.org/10.1155/2019/5214821
Hello Ladan Kalani
Please refer to Evaluation of Separation section given in the link below. It may help.
http://www.biorad.com/en-us/applications-technologies/performing-protein-electrophoresis
Best.
Malcolm Nobre and Shail K Chaube Thank you so much! I suspect it is the gel that is problematic as I am seeing the smear even on wells that did not contain an abundance of this protein. I did use an old SDS and reused sample buffer.
I suspected that issue,. Since it was not pointed out in your details , I did not mentioned this issue.
As you now said it, use of old SDS and used sample buffer might cause the problem you are facing.
I would suggest to use freshly prepared buffers to rule out this possibility.
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