Hello
I am using OxPhos Rodent WB Antibody for mitochondrial protein expression study from muscle sample. But I faced difficulty for getting 5 different mitochondrial complexes after using this AB. I have seen whole protein in the blot and it was really looking good and proper differentiate in the blot but after adding primary and secondary AB all 4 complex band disappeared. Only complex V is present in the blot. I used 10% gel and 20% gel, boiled sample at 95oC for 5 mins, even without boiled also and 5% non fat milk for blocking. Using primary AB dilution factor 1:3000 and secondary ratio is 1:10000.
Would anyone please give me any suggestion regarding this matter?
Thank you
Hey, I understand your concern regarding the difficulty in detecting 5 different mitochondrial complexes after using the OxPhos Rodent WB Antibody for your mitochondrial protein expression study from muscle samples. It's good to hear that you could see the whole protein in the blot and that it looked proper and differentiated. It's possible that the primary and secondary antibody concentrations you are using may not be optimized for detecting all 5 mitochondrial complexes. You can try different dilution factors for the primary and secondary antibodies, as well as trying out different blocking buffers or changing the transfer conditions. Another suggestion is to try running the gel longer or using a gradient gel to improve separation of the protein complexes. You could also try different antibodies specific to each complex to confirm the results. I hope this helps and good luck with your experiments!
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