Hello.
This is the membrane I got after WB and chemiluminescence detection (68 seconds) on my human neuroblastoma SH-SY5Y cells extracted in RIPA+anti-phosphatase/protease (anti-SIRT1, Abcam, ab12193, dil. 1:500 + HRP-conjugated anti-rabbit, Vector PI1000, dil. 1:1000).
Abcam reports that the Ab "detects a band of approximately 110 kDa (predicted molecular weight: 82 kDa)..." along with "...a band of approximately 110 kDa (110-121 kDa) which is likely to be due to post translational glycosylation" (http://www.abcam.com/sirt1-antibody-e104-ab32441.html?pageNumber=2&productWallTab=ShowAll)
Does anyone know what band should I consider as the "right" one? May that be the one at ~135 KDa? What kind of highly-expressed forms am I detecting at lower KDa?
Has anyone faced this problem before?
Thank You very much in advance.
Stefano Falone
Dept. of Life, Health and Environmental Sciences
University of L'Aquila
it is look you detect your pull-down abs much better than your sirt1. i would suggest to try your ab first on a simple blott without pulldown. how it works, what is the best signal, etc. my experience, sirt1 is low in cells, but i was able to detect from a well of 6-well plates (HeLa) a resonable amount. if you use the same ab for pull-down and detection, there is some trick to resolve the presence of excess IgGs (from the same species) on your blott but i only read about that.
I'm not sure I understand your reply. my secondary Ab is an anti-rabbit antibody and it should not be able to cross-react with human IgGs. Why do you think that the extra bands I detect are likely due to unspecific interaction between human IgGs and my secondary Ab?
you absolutly wright, i misunderstood heavily your question. however, your pattern is still similar to an IgG pattern, likely an IgG-like receptor, or some FCS residues from TC medium (a heavy chain about 60 kDa and a light chain about 20 kDa).Is your secondary cross-reacted against human serum?
My secondary Ab is described here:
http://vectorlabs.com/peroxidase-goat-anti-rabbit-igg-antibody.html
Its cross-reaction with human IgGs is certified to be less that 1%.
i check the vendor site. they suggest to use some serum from experimental host in sec ab diluent if you detect cross-reactivity. Mightbe this is not that but you still can try it.
Hi, working with rat/mice tissues the antibody Sirt1 (ab110304) gives the band at 120 KDa.
Thanks so much, Maria-Nieves. I checked your Ab code and it says that it is a mouse monoclonal to SIRT1. Mine is a rabbit polyclonal to SIRT1. Maybe, for this specific protein I should buy a monoclonal Ab. Do you have extra bands with yours?
Hi, I have used it only in mouse tissues and it works very well. You can find one or two bands depending on the tissue: two (heart) and one (skeletal muscle and liver). The antibody is very clean and you can see clearly the band(s) at 120 kDa. Good luck!
Hi Stefano Falone, I was just going though this discussion. I'm curious to know finally which one you considered ? or what did you do ? Because I am right now having the same problem with SIRT1 (from Santa Cruze)
Hi Kowshik K.,
here You can find attached the details of a WB that was carried out on HUVECs-derived total proteins (20 ug).
Please, feel free to contact me if You need anything else.
Regards
Stefano
Update.
We changed primary antibody and chose Abcam anti-SIRT1 (cat. no. ab7343).
Well, that's much better!
Stefano
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