Hi,
I'm wondering why the first blot (_170858) shows such wavy bands. Is it an issue with the gel or the transfer? Also the lower blot in the second image (_172622) shows a decreasing signal in the loading control (alpha tubulin) from left to right. The ladder for that blot also did not transfer consistently on the left compared to the right. What could cause the unevenness?
I'm using Bolt® 4-12% Bis-Tris Plus Gel from Invitrogen for both, running for 3 hrs in MOPS-SDS buffer. I use wet transfer using a Bio Rad Mini Trans-Blot Cell onto a PVDF membrane.
Thank you for your help.
Hello, the weavy bands are probably due to your gel. Try to make it properly (avoid the air bubbles). The unevenness of your loading control mean that your loading should be modified for the inequal bands or you have to pay more attention to all parts of your membrane when you do the different steps especially the primary and secondary antibodies blotting (maybe they couldn't access these parts properly). Good luck
Hi Iskander,
Thank you for your suggestions. The gels are pre-made from Invitrogen, so I'll try to see if there are any differences among different gels in the batch. I'll definitely pay extra attention to loading and antibody incubation. I'm wondering if the transfer apparatus can also contribute to unevenness since it's often the second of 2 gels that have this gradient of tubulin signal strength.
Thank you again.
Iskander Madhi is right ! This could be due to either your poor transfer (air bubbles) or poor gel quality. May be your first or second antibody did not reach equally! You may use GAPDH as a loading control. I had also experienced this problem with tubulin. Best wishes
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