Does anyone have an idea as to why two commercial ABs detect ATF4/CREB2 at different masses in identical samples, and which of these might be more trustworthy?
I've used both Cell Signalling 11815 and Santa Cruz 200 to detect ATF4 in mouse cells and tissues.
CS-11815 is much cleaner, and is a monoclonal from Cell Signalling, which I trust to be better QC'd than many polyclonals; so I've favored it in my work. However, CS-11815 detects a band with a mass of ~49 kDa, while the expected molecular weight of ATF4 is ~38 kDa.
In contrast, SC-200 is a very dirty polyclonal antibody, but it seems to detect ATF4 as both a ~49 kDa band and a ~38 kDa band.
I'm unaware of any long ATF4 splice variants or PTMs that might be responsible for this difference. Also, I can't get a truly appropriate ATF4 negative control (ATF4 KO tissue), so we can't even really be sure any of the bands detected really are ATF4. The controls shown for SC-200 are merely from mice expected to express high or low amounts of ATF4 based on previous work (controls not included for CS-11815, but they'd show similar results at ~49 kDa - High intensity in the positive and low in the negative).
Using ATF4 KD or KO cells as a control might work, but it wouldn't recapitulate the background bands we see in tissues, making such controls rather imperfect.
Both of these ABs have been used in numerous previous publications, so I can't just dismiss one or the other as being junk without a good justification.
Hi Brian. I've worked extensively with both the SCBT and Cell Signaling anti-ATF4 Abs in question and had similar experiences. For blotting of whole cell extracts, I think the Cell Signaling antibody is more reliable. The band recognized by the mAb D4B8 is depleted by siRNA whereas several cross-reacting bands persist with SC-200. The migration difference between the observed vs predicted molecular weight of ATF4 has something to do with the primary sequence (possibly incomplete denaturation by SDS) since it is observed in both mammalian cell lysates and with recombinant ATF4 (E. coli expression). So the 49kDa band is the correct one. If you want to use SC-200 for blotting, I'd recommend biochemical fractionation to isolate nuclei prior to blotting; this eliminates most of the background bands.
Dr. Cohen, thanks for your reply and insight. I dug through my notes from some of my cell culture work, and I'm inclined to believe you. I found that treating skin fibroblasts derived from adult mice (Mouse Adult Fibroblasts, MAFs), with either Thapsigargin or Cadmium induced expression of a ~50 kDa band detected by CS-11815. I didn't test SC-200 very much in these experiment, but serial-blotting with 11815 then 200 didn't reveal a detectable increase in a ~38 kDa band.
The blot to the right shows the positive and negative controls (Liver from Snell Control and Dwarf mice), that were omitted from the original post.
Hopefully this will be enough to convince my mentor that the apparent mass of ATF4 is indeed 49 kDa, though it may be prudent to test SC-200 in a standalone blot with these drug treated samples.
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