Dear Community, I recently performed a Western Blot to test whether the mitochondria marker ATP5A1 is in the experimental cells or not. However, the results did not show up at the expected MW . I would like to ask what happens in this case and my sample 1 even didn't have band with beta-actin marker? Would you like to give me some advice ?Thank you so much .
Hi Phung! I would suggest using this link on biorad western blot troubleshooting. I have not had the exact same experience. However, when I need to troubleshoot my western blot I always use this link.
https://www.bio-rad.com/en-us/applications-technologies/troubleshooting-western-blots-with-western-blot-doctor?ID=MIW4HR15
In addition, I recently have had trouble with Beta-actin as my housekeeping gene. It was not working well with my protein samples. Therefore, my postdoc suggested using GAPDH (cell signaling) as an alternative antibody. thankfully, using GAPDH worked with my samples and allowed for me to observe even bands. I hope this information will be useful. Good luck!
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