In addition to the “usual” protein standards, Bio-Rad Laboratories manufactures the Precision Plus Protein WesternC standards which contains a Strep-tag affinity peptide that has intrisic binding affinity for both native streptavidin or StrepTactin and can be “lit” up with the appropriate chemiluminescent stubstrate during your western blot chemiluminescent development. However, when using a fluorescent-based stain for visualization, an unstained standard must be used. This holds true for both colorometric and chemiluminescent protein (or protein tag) visualization. In western blotting, prestained standards are the standards of choice since they can be used for both MW estimation as well as for determining the efficiency of protein transfer. Prestained standards are ideal for monitoring the progress of electrophoresis. MW estimation can be determined by plotting unknowns agains a standard curve which is generated by calculating the relative mobility of each protein standard and plotting the mobility of each band against the log10 of its MW. They are also required when a fluorescent dye is used to stain the gel or blot to visualize total protein. Unstained standards are unencumbered by protein dyes and therefore give the most accurate electrophoretic mobility pattern. When using gel electrophoresis for molecular weight (MW) determination, unstained standards are the ladders of choice. The guide will be broken down into both gel electrophoresis and western blotting applications. The information was distilled from an article published in issue 127 of Bio-Rad Laboratories’ BioRadiations Magazine and can be found in its entirety on page 11 when clicking on this link. In the following post, we will share with you a practical guide for selecting appropriate protein standards. So I don't understand how the protein could either come out the other side, or not go in at all, but the ladder be fine.Apat 2:16 pm Canadian Biotechnologist 1 comment Would one of the two observations listed above suggest something that would allow the pre-stained ladder to transfer successfully, but not the protein? The ladder transfered evenly from 250 all the way down to 15 kD. So there is no protein, but the curious thing is the pre-stained ladder came out fine. pvdf thread in this forum that not soaking in water long enough can prevent protein transfer in PVDF)Ģ) His transfer chamber, when put at the same voltage I normally run (30v) went up to something like 260 mA which is about 4 times higher than the normal mA I see with my mini-transfer chamber. I told him to pre-wet in methanol, which we did, but instead of soaking in water for 5 minutes as per the instructions, he just rinsed and put in the transfer sandwich. ![]() When he was putting together the transfer, he dunked the pvdf directly in water and acted surprised when it didnt hydrate. Two suspicious things:ġ) This professor always used nitrocellulose and we use PVDF (hybond-P). In fact, except for the sample preparation in sample buffer, he pretty much did everything. I ran the big gel with professor who does them a lot. However, I wanted to get more resolution so for this particular western I had run a "big" gel. In fact, I had run these exact samples already twice and it worked fine. ![]() I run mini-gels all the time and never have problems. I encountered something strange the other day with my western blot:Īfter transfer, the pre-stained ladder (bio-rad) showed up fine, but there was no protein whatsoever on the blot stained with Ponceau.
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