Transfer of proteins with high isoelectric point (pI) Also, use Ponceau S to make sure proteins were transferred from the gel efficiently onto the membrane and stain the gel with Coomassie Brilliant Blue.ħ. It is recommended to use a PVDF membrane instead of nitrocellulose for high MW proteins. Also, it may be necessary to eliminate methanol from the transfer buffer as methanol inhibits elution of high MW proteins out of the gel matrix. Please note that high MW proteins (above 100kDa) generally transfer more efficiently overnight in a wet transfer apparatus with the addition of SDS in the transfer buffer. Hence, for smaller proteins and peptides, alternative methods such as the one developed by Schägger and von Jagow (1987) may be necessary. Note that the standard SDS-PAGE using Laemmli glycine-based buffers has a minimum resolution of approximately 16kDa. Especially for complex protein mixtures that are spanning a high MW range, you may need to deploy gradient gels for best resolution. To get a good resolution, lower and lower concentrations of PA should be used as the MW of target proteins are getting higher and higher. Standard SDS-PAGE gels range from 7 to 15% PA. Adapt gel to match the requirements of your target proteinĬhoose the type of gel and its polyacrylamide (PA) content depending on the MW of your target protein and the required accuracy of the MW determination. Be aware that due to retaining its three-dimensional structure, your target protein will show a higher apparent molecular weight (MW) in blue/native gels than under standard denaturing conditions.ĥ. In those instances, a blue/native gel needs to be run. In some cases, this treatment may, however, disrupt the conformation of a three-dimensional epitope that is recognized by a monoclonal antibody. The standard SDS-PAGE for subsequent Western blot analysis is being done under reducing and denaturing conditions. In those instances, you should consider incubating your samples with sample buffer at 60 ☌ for one hour instead of boiling for five minutes. It will do no harm to your samples and you will be on the safe side.īe aware that highly glycosylated or hydrophobic proteins such as multi-drug resistance (MDR) proteins may precipitate upon boiling. If you are uncertain whether your sample buffer still contains enough reducing agent (NOTE: its effective concentration will decrease with time!), simply add extra. dithiothreitol (DTT) or β‑mercaptoethanol) in your sample buffer will need to be high enough to effectively break-up all proteinaceous disulfide bonds. If gels are run under denaturing conditions, the actual concentration in reducing agent (e.g. In general, samples should be boiled five minutes in complete Laemmli sample buffer, centrifuged and only the supernatant should be used for SDS-PAGE analysis. Be aware that commonly used protease inhibitors such as phenylmethanesulfonylfluoride (PMSF) may have short half-lives and need to be re-supplemented with time. Samples should always be prepared quickly, cooled on ice and pan-protease inhibitors added to avoid degradation of proteins. We know that Western Bloting has nothing to do with cowboys (thanks Sir Southern!), but this is a nice picture
0 Comments
Leave a Reply. |