Hi everyone, I need your help,
I was performing western blotting, and during the detection, a wavy band appeared.
Could you please give me some advice on what to do with the wavy bands?
Thank you for any advice.
You could try desalting the samples or just loading less sample on the gel
Do you make your gels or purchase them? If you make them, give the combs and plates a really good wash.
For either type, rinse out the wells really well before loading (rinse with the running buffer). The goal is to get rid of any small gel fragments or unpolymerized gel.
Most of the bands look great, it might be something specific to that protein in particular.
It seems like a lot of bands post-secondary antibody. What are you blotting for?
Amy Klocko
I am making my own gels. I always wash the combs and plates before using. I also do what you mentioned about rinsing out the wells. As you said, maybe there is a problem with the secondary antibody. Recently, I have tried different dilutions and it looks better :D
Thank you for your advice.
Minuet Barragán
Hi, I did not try desalting, but we found out that there was a problem with the polymerization of the gel. For running, we first use 55V for 20 min, and then we use 150V for approximately 1 hour and 30 minutes (it depends on how fast it goes). For transfer, we use 12V, 1A for 60 min.
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