Question (71229) | Anti-mtTFA antibody [18G102B2E11] - Mitochondrial Marker (ab119684)

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Question

The samples are prepared from schwann cells and DRG neurons and pancreatic cancer cell lines (one human, one rat origin, one mouse origin ) we chose the antibodies according to the reactivity mentioned on the abcam data sheet and the lysates were prepared using a non denaturing lysis buffer, aliquoted and stored at -80 degrees centigrade. I use 10% and 12.5% acrylamide gels depending on the molecular weight of the protein. initially 40 ug protein was loaded and the gels were run at 70 volts and transfered on to PVDF membrane (ice cold). All buffers were prepared as suggested in the western blot guide by Abcam. 5% milk was used to block the membrane, washed thrice with TBST and/ or TBS. Incubated with respective antibodies (initially 1: 1000 dilution) overnight in cold room with agitation. The blots were added with secondary antibody the following day (secondary antibody from abcam (ab97023 and ab97051 which were aliquoted and stored at -20 degrees centigrade) incubated for 1 hour at room temperature and then washed thrice with TBS/and or TBST. probed with chemiluminescent solution from Biorad. Bands were not detected. We tried loading 50ug protein (we cannot load more owing to the limited volumes of sample we have and the nature of the project) and then tried using 1:500 and 1:250 dilutions, but still the antibodies were not working. We have been using this protocol and works fine with all the other abcam antibodies. Please let me know if I need to elaborate on any of these steps.

Answer


Upon reviewing your protocol, it is troubling to me that all 4 of these antibodies are not working. While it is conceivable that all four antibodies are not performing properly, typically when multiple antibodies are not working it is an issue with sample preparation, membrane transfer, or the detection substrate. In your case, I think the following troubleshooting steps are the most worth your while attempting, with tip 1 being the first thing you try since it is the most likely to make the targets recognizable by the antibodies:
1. You mentioned your samples were non-denatured. ab2571, ab32124, and ab81212 were all raised against synthetic peptides and will require linear proteins in order to recognize their epitopes. Thus, your samples should be both reduced and denatured (SDS and beta-mercaptoethanol or DTT) and boiled for 5 minutes at 100C prior to freezing at -20C. RIPA buffer is a good choice for mitochondrial and membrane proteins such as these.
2. To enrich the target proteins, have you considered a mitochondria isolation kit such as ab110170 (https://www.abcam.com/Mitochondria-Isolation-Kit-for-Cultured-Cells-ab110170.html)?
3. Have you insured that the proteins are being transferred with a reversible stain such as Ponceau S? Since you are using PVDF, make sure to pre-soak the membrane in MeOH, then in transfer buffer.
4. If you are including milk in your antibody dilutions try removing milk or reducing to 0.5% milk for the antibody dilutions only.
Further troubleshooting tips, including no signal specific tips, are included through the link below:
https://www.abcam.com/index.html?pageconfig=resource&rid=11352
Please let me know the results of troubleshooting, especially step 1. I look forward to your reply and sincerely hope this information helps.

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