Dave Weaver generated new PER2 antibodies can be used ICC. This aniboty is commercially available from Millipore. Please see competing interests section in their publication (they discribed product number in competing interests section in their paper).

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0035938;jsessionid=7DE6AB4D242303D713613434724AF2B1

Have you tried contacting the manufacturer? I do not use the specific antibody, but from my experience, if the antibody was stored correctly (aliquoted, the right temp etc.) and you are sure you performed troubleshooting for the western protocol (ponceau staining, validating running and blotting conditions) , then I suggest you try the company for further assistance.

Hi

Thank you so much for your response!

Yes, we had contacted with the company and got the completely new antibody for free.

Totally we examined 4 tubes (2 tubes of 869900A1.7 and 2 tubes of 869900A1.10) and some tubes were new one.

Now the company can supply only #869900A1.10, so we cannot try other Lot#.

We tried both DAB and fluorescence immunohistochemistry.

In our lab, we have been used the antibody for 5 years, and could get the good signal.

Anyway, thank you for your help.

Hi Takashi

We were using one from Kerafast that was great but then demand exceeded supply and they ran out. They may get more at some point. We tried Millipore but we had inconsistent results - it seems that whether the antibody works varies from lot to lot. Sigma will let you reseve as much as you need for your project but we have been unsuccessful with using their Per2 antibody. If you find a solution, please let me know because we are having the same problem.

Thanks,

Karen

Has anything changed in the protocol? If you detect signal by immunohistochemistry with the same antibody I would would consider looking into other factors that might affect your assay.

I am sorry I cannot be of further assistance.

If you would like to try antibodies from other suppliers we can help you locate them. Just search here: http://www.linscottsdirectory.com/search/antibodies for PER2, and select "Mouse" and "Immunohistochemistry" from the Reactive Species and Applications pull down menus. You will see there are a number of companies offering such Abs, which you might want to check with.

Dave Weaver generated new PER2 antibodies can be used ICC. This aniboty is commercially available from Millipore. Please see competing interests section in their publication (they discribed product number in competing interests section in their paper).

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0035938;jsessionid=7DE6AB4D242303D713613434724AF2B1

Hi Takashi,

We had the same problem when they move to a new bleed and had to find a new supplier. ADI's tech support were no help either.... Try Santa Cruz's Per2 antibodies, we use sc-7729, it's a good alternative.

Shin is also correct, Millipore carries David Weaver's Per2 antibody that also works very well in the rat model, although we haven't tested it yet in the mouse model though.

Hi Karen,

Thank you for your response. I speculate Millipore antibody is AB2202 which is made by David Weaver (Plos one 2012). This is one of the candidates because they showed beautiful pictures. The small issue is that in the web site they only reccomend Western blotting. Also thank you for the Lot to Lot information. We cannot know it until we try several tubes. Now that I got a lot of infromation and will let you know if an antibody works well.

Thank you.

Takashi

Hi Maayan,

We used the same protocol, examined 4 tubes, and tried both DAB and fluorescence.

Other immunohistochemisties are working well.

That is why I doubted the antibody.

Thank you for your comments.

Takashi

Hi Russell,

Thank you for the information. I could detect many PER2 antibodies. This is useful not only PER2 but also other antibodies.

Thanks,

Takashi

Hi Shin,

Thank you for your reply. Millipore antibody (AB2202) is one of the candidates, although the web site suggests only Western blotting. In the manuscript (Plos one 2012), they showed beautiful pictures. I will consider it.

Thank you.

Takashi

Hi Barry,

Thank you for your comments. You and Dr. Shimon Amir always show beautiful PER2 staining. I believe your group had used ADI antibody and that's why we selected it. I alos cheked recnetly your group used Santa Cruz antibody. It looks like Millipore and Santa Cruz are excellent. I will talk with my boss. Antibodies are expensive.

Thank you for your help.

Takashi

Hi Takashi,

We have tried the Millipore antibody (AB2202) as well as one of the original bleeds of this Per2 from Dr. David Weaver, both work well for IHC. The Santa Cruz antibody is very good as well in an IHC paradigm, it's really a matter of preference. Some companies will supply trial sizes for much less the cost, so that might be a way to go...

Hi Barry,

It's great if we can try trial sizes. I appreciate it.

Thank you.

Takashi

Dear all, interesting discussion on PER2 antibodies!

Did someone try to stain other brain regions with PER1 or PER2 antibodies? I ask this because in mouse hippocampus Western blots the David Weaver Millipore anti-PER1 in my hands makes multiple signals with the ones in the expected size range 130-140 kDa not among the strongest. Under preabsorption the signal are almost all gone. I would be grateful for any hints on how to solve this problem!

Hi Erik,

We saw multiple bands in all our WB for Per1/Per2 (AB2201/AB2202) and could not get rid of it. The data-sheets for the antibody also show multiple bands, so I don't know if you can get rid of it altogether. Westerns are not my forté, but interestingly the IHC staining for Per2 is nuclear in some regions and cytoplasmic in others, could this account for the band differences?

Hi Barry,

it is well possible that the extrabands are extranuclear. Using nuclear extracts instead of whole-cell extracts may solve this problem. I will try it (next year).

Hi Erik,

Our lab published Hippocampus PER2 paper several yeas ago.

http://www.ncbi.nlm.nih.gov/pubmed/19570032

I didn't perform Western for the manuscript, but our lab member could detect a signle PER2 band with Alpha diagnostic antibody (PER21-S).

Recently I tried PER1 (Alpha diagnostic, PER11-A, Thermo scientific, PA1-524) and PER2 (Alpha diagnostic, PER21-S, PER21-A) in Striatum, but I couldn't detect the single band.

Thanks Takashi,

I was aware of your PER2/Hippocampus paper since my collegues here have done similar work on Per1-KO mice (Jilg et al., 2010 Hippocampus), but the PER1 antibody used for this study is nearly gone and so far attempts to replicate it have failed. I will try to use nuclear extracts as said above and hope that the number and MW of the signals appearing makes sense. I saw that Nikolas Cermakians lab just published a paper on PER1 regulation and the blots there are similar (although a bit better) to those I made on whole-cell or whole tissue extracts. In pineal and liver of hamster the ADI PER1 and PER2 antisera worked well in the past (Maronde et al., 2007) but in mouse extra SCN tissue I had lots of signals also with these and many other commercial PER1 and PER2 antisera.

Anyway, I thank you all for the nice hints and helpful comments and promise to keep you informed on the issue of PER1-Western is in mouse extra-SCN tissue!