Anti-VDAC1 / Porin antibody [20B12AF2] - Mitochondrial Loading Control (ab14734)

Thank you for you rapid answer. Concerning the COX5A, it is detected by the secondary antibody from Jakcson Lab. I blotted on the same membrane against COX5 and VDAC and only got signal for COX5A using secondary antibody from Jackson Lab. So it is efficient against IgG2a. We have not pay for this antibody yet. But we will order a different mouse VDAC antibody, for instance ab14734, as soon as we know that we do not have to pay for ab61273.

ANSWER:

Thank you for the information regarding the efficacy of the secondary antibody.
I will be happy to send ab14734 as a free-of-charge replacement for ab61273 now, if you like. You will still need to pay for ab61273, if you have not done so already. Shipping will be free for the replacement.

WB fine, ICC not used mouse T cells and KO cells WB: sees 1 band at right MW in WT, in KO sees no band as expected Ab: 1/500 ICC: 1/100 - 1/2000 for 1h and o/n problem: WT and KO gives same staining everywhere, also different dilutions and staining times shows no difference in staining (staining everywhere) fix w/ PFA block w/ goat serum secondary: OK, alone (no primary) -no signal

ANSWER:

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products. I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number xxx forab15895. To check the status of the order please contact our Customer Service team and reference this number. Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know. I wish you the best of luck with your research.

I'm using this antibody in WB and I'm seeing a consistent band intensity across lanes at 37kDa.  However, I'm seeing varying intensity of the bands at 50 and 75 kDa so I'm not convinced that this is from secondary binding.

ANSWER:

Thank you for your inquiry and I apologize for the delay in this reply. 

I heard back from the testing lab and they did confirm that the extra bands at 75 and 50 kDa are related to the samples. When using tissue samples, especially mouse tissues, usually you cannot exclude all the blood when preparing the sample.  The blood contains mouse IgG, and when the sample is run on a gel, the IgG (heavy chain or light chain ) also runs into the gel and is distributed at 75 kDa (one set of heavy chain/light chain) or 50 kDa (just the  heavy chain).  These bands can be recognized directly by the secondary goat anti mouse antibody. When using whole cell samples e.g. HepG2, you should not see this extra band.   I cannot recall whether or not you were using mouse tissues or cells, but you may see varying intensity in these bands due to various amounts of blood/mouse IgG in the sample.

The other way to avoid the extra band is to use TrueBlot® as your secondary antibody, which will only detect native IgG but not denatured IgG.

I hope this information helps.  Please contact us with any other questions.    

Thanks for your rapid response regarding the VDAC antibody. The band I am detecting is approximately 31kDa, whereas the predicted molecular weight on the data sheet was 39kDa? If it is the phosphorylated and unphosphorylated porin, then would you suggest analysing the top or bottom band or both together? Blocking buffer comes from Odyssey.

ANSWER:

Regarding the molecular weight of VDAC1, the precursor of the protein, before any post-translational modifications such as phosphorylation, is predicted to be 30.8 kDa for mitochondrial VDAC1.

VDAC1 is present also in the plasma membrane. In mice, this plasma membrane VDAC1 is 32.3 kDa. This might account for the two bands you are seeing near 31 kDa, but I have not been able to find information about the molecular weight of a human plasma membrane -specific VDAC1. A description of the two mouse isoforms can be found in the UniProt entry at the following link:

http://www.uniprot.org/uniprot/Q60932

Demonstrating that the two bands represent proteins specific for either the mitochondrial membrane or the plasma membrane would require preparing cell fractions, separating plasma membrane from mitochodria and running the two fractions in separate lanes of a gel (with a third lane for a whole-cell lysate for comparison).

We have a kit for preparing a mitochondrial fraction, if you are interested in trying this. The catalogue ID is ab109719:

Click here (or use the following: http://www.abcam.com/index.html?datasheet=109719).

To check for the possibility that the heavier band represents phosphorylated VDAC1 also requires some work. What some people will do to demonstrate that a blot band is a phosphorylated protein is treat the sample with a phosphatase to de-phosphorylate, then run it next o an un-treated sample for comparison. The band representing the phosphorylated protein should dissappear and the protein will now migrate to join the rest of the un-phosphorylated VDAC1. But I think the cell fractionation should be done first, to check for sub-cellular fraction-specific isoforms.

Regarding the sizes of the bands in the blots on the datasheet, I do not have an explanation for why the protein in thsoe samples migrates to 37 and 39 kDa. For comparison, the datasheet of another VDAC1 antibody, ab47104, has a blot similar to what you are getting, with two bands near 31 kDa.

Click here (or use the following: http://www.abcam.com/index.html?datasheet=47104).

If you simply want to try a different antibody, rather than trying to understand your results with ab14734, I will be happy to send a replacement with a different antibody, though if your sample does in fact contain these two isoforms, you are likely to see the same two bands. I suggest ab15895.

Click here (or use the following: http://www.abcam.com/index.html?datasheet=15895).

Please let me know what you would like to do.

Here's the answers to your questions:   1. I used blocking overnight at 4 C in 5% non fat milk 2. The wash buffer is TBS with Tween 20 (T/TBS) 3. I used BSA in the porin-containig T/TBS buffer instead of milk since BSA is usually recommended for monoclonal antibodies; the antibody was diluted to 1 ug/ml 4. Yes, I tried both 1h at room temperature and overnight at 4 C with similar results As for the ab16056 (anti-COX IV) antibody I can not use it as loading control since we observe effects on respiratory chain proteins in our system.  

ANSWER:

Thank you for your reply. I was just talking to the people from the lab and they have told me that ab14734 may not be suitable for Blue Native applications. I do apologize for the misinformation that originally received. After having talked to them, if you could send the entire protocol that you have used (including buffers) we can see if we can recommend any changes that will increase the antibodies performance. The only product that we have in our catalogue that is guaranteed to work in Blue Native applications the ab110412, the MitoProfile® Total OXPHOS Blue Native WB Antibody Cocktail, which you have already said is not suitable. I am sorry once again over the confusion, please let me know if there is anything else I can do.