BioLegend will not be held responsible for patent infringement or other violations that may occur with the use of our products. Kyle Cottrell, Sergej Djuranovic 2016.(PubMed link indicates BioLegend citation) For consistent results, please use TBS/T buffer for Western blotting that contains 0.15 M NaCl as indicated in the BioLegend recommended protocol. The binding of this antibody to its target is sensitive to salt concentration. The optimal dilution should be determined by titration for each individual assay of interest.Ģ5 µl and 100 µl of Direct-Blot™ HRP antibody can be used for approximately 5 and 20 Western blots, respectively, at the recommended concentration/dilution. For Western blotting, the suggested dilution is 1:1000-1:2000. Upon receipt, the antibody solution should be stored undiluted at -20☌, and protected from prolonged exposure to light.Įach lot of this antibody is quality control tested by Western blotting. Lot-specific (to obtain lot-specific concentration, please enter the lot number in our Concentration and Expiration Lookup or Certificate of Analysis online tools.) The antibody was purified by affinity chromatography and conjugated with HRP under optimal conditions. 2-fold) without careful consideration, so for instance your lanes 4 and 5 (not including ladder) have faint actin, but the POI is more or less consistent with the surrounding lanes - meaning that in 4 & 5, you have much more POI than in those other lanes, but it takes some thought to realize this.This antibody is provided in 50% glycerol in aqueous buffered solutions with preservatives. Having a plot of normalized results is all well and good, but the eye is not very good at determining changes of less than about 50% (i.e. However, for publication, you may have a harder time convincing reviewers that your results represent real data if the actin bands are so variable. It are the best descriptions of the conditions needed that I have come across. Please read the PDF here from Li-Cor on western blot normalization. Please note that this image would not probably not meet most publication requirements (in my opinion). You can not compare results across more than one blot without running inter-blot comparison controls! For normalization you generally need to have loaded less than 10 ug of protein, have not over-exposed any of the bands, and have them all loaded on the same gel and blot for comparison. The long and the short of it is that your actin bands do not need to be consistent so long as you can normalize and have met the quite stringent conditions for normalization to be effective. Or would I need to rerun this sample again? However, I noticed that my actin bands are not consistent (some are fainter or darker than others).ĭo the actin bands have to be the same darkness for each sample? Could I still use these results and instead do a normalization? Top band is actin and bottom one is my protein of interest I have attached a photo of my membrane down below. I probed my membrane for my protein of interest and a housekeping protein (actin). In addition to the tissue, I also loaded the same volume of Laemelli buffer as tissue and I used RIPA buffer to make the volumes of each sample constant. Also, the tissues have undergone multiple freeze and thaw cycles. However, the BCA analysis was performed years ago (approximately 4-5 years ago). The volumes were determined via BCA analysis. I loaded the samples on the gel to ensure that for each tissue, I had a constant mass of protein (30 ug). I have been running Western Blots on rat brain tissues from rats that have been subject to neurological disorders.
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