high mass standard protein ladder western blot

For SDS-PAGE followed by western blotting, use pre-stained MW markers. For SDS-PAGE use either an unstained MW standards or pre-stained MW marker. Prepare MW standards for electrophoresis.Sample protein concentration should be sufficiently high eg.: final protein concentrations from 1 µg–500 µg depending on protein type and detection method. Any other protein samples: transfer to clean pre-labeled microcentrifuge tubes and mixed with an equal volume of 2X Sample Buffer with 0.55M BME.Label microcentrifuge tubes with sample description, volume and concentration of lysate. Add β-mercaptoethanol (BME) to a final concentration of 0.55M, i.e. If using a pre-prepared lysate (already in sample buffer), thaw lysate and transfer 25 µL of lysate to a clean pre-labeled microcentrifuge tube. New England Biolabs offers a selection of highly pure protein standards.A mixture of these proteins are called protein standards or protein molecular weight markers. To be able to estimate the MW of proteins on the SDS-PAGE, proteins of known MW need to be run simultaneously on the gel. In PAGE the relative migration distance of a protein (Rf) is negatively proportional to the log of its MW. Some caution has to be put on intrinsic strong negative or strong positively charged proteins because SDS may bind to them differently and their migration in the gel may not be at the expected MW. Because the charge-to-mass ratio is nearly the same among SDS-denatured proteins, the final separation of proteins is almost entirely dependent on the differences in relative molecular weight (MW) of polypeptides. The negative charges on SDS destroy most of the secondary and tertiary structures of proteins and are strongly attracted toward the anode in an electric field. SDS is an anionic detergent and is used to denature the proteins. SDS-PAGE allows an estimation of the purity of protein samples.

High mass standard protein ladder western blot