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1. The left 6 lanes represent 6 Normal pigs. They were healthy and untreated with drugs. Themiddle6 lanes represent 6 pigs with Renal artery stenosis (RAS), but untreated with drugs. 6 lanes on the right represent RAS pigs treated with drugs. The drug was thought to preserve the kidney function. I do not know the expression level of angio-1 in normal pigs. We want to see if there is any change in these animals.Basically, there is no negative control. 2. I did not test the residual signal frompreviousblot. Theangiopoietin-1 (both ab8451 and ab95230) was tested after b-actin but before the others. That strong band around 44 kDa might represent residual signal from b-actin in picture of ab95230. But the other bands were not likely from residual signal.
Asked on Jan 05 2012
Thanks again for the additional data. I think there are a few confounding factors here that make the western blot data difficult to interpret confidentally: 1. I agree with you that the intense 40kDa band is likelyb-actin in both blots, but we can not be certain. 2. There also may be bleed-through from the first anti-angiopoietin blot to the second angiopoietin blot. 3. Pig angtiopoietin-1 has technically not been tested previously with either ab8451 or ab95230, so the specificity of these antibodies with respect to pig samples is not known. 4. The expression pattern ofangtiopoietin-1 in pig kidney tissue, including any alternate forms or glycosylated forms, are not known, at least based on my brief literature search. You may consider treating your samples with or without PNGase-F, an endoglycosylase which would remove complex oligosaccharides from N-linked glycoproteins. If treated samples show a band with a smaller molecular weight, this suggests the detected protein was gylcosylated and likely angtiopoietin-1. I look forward to hearing if these suggestions prove useful.
Answered on Jan 05 2012