-SDS-PAGE is only for rough estimation but not for precise determination of protein molecular weight (MW), due to variation in amino acid compositions of proteins and varied ionic species in the PAGE buﬀer. -Prestained protein markers are required to be calibrated in various running conditions for more precise MW estimation. -ExcelBand™ PM2700 and PM5200 have molecular weight range of ~5kDa to 245kDa, showing consistent migration curves when calibrated against natural proteins.
Protein molecular weight markers or protein markers (PM) are a staple tool in most molecular biology laboratories. In choosing the appropriate marker, researchers contemplate on the precision and sizes oﬀered by the product. Other factors that concern researchers are the convenience in determining the sizes and the price of the product. In this article we would like to discuss the issue of precision when running SDS-PAGE with your PMs as well as your proteins.
The determination of the molecular weight of a sample protein is done using Sodium Dodecyl Sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) method. The protein is supposed to be linearized (denatured) and imparted with negative charges by SDS, causing the protein to migrate towards the cathode. The negatively charged SDS is supposed to distribute evenly to the protein which enables the protein to migrate and be separated during electrophoresis, with migrating speed largely based on the size of proteins. However, the native charge of the amino acids (amino acids are zwitterion) of the
protein also contribute to the overall charge of the protein in a given pH. Table 1 shows the variation of amino acids relative to the pH of the environment. Furthermore, the molecular weight of membrane proteins, which often contain hydrophobic trans-membrane domain or hydrophobic proteins, are harder to determine using SDS-PAGE method due to the higher variable binding ratio to SDS . In many cases the migration pattern of these proteins are widely misrepresented in SDS-PAGE analysis as shown in Figure 1.
Fig 1. The complexity of the protein intrinsic properties which present higher variability in their migration property, can result in a wildly misrepresented molecular weight while using standard SDS-PAGE analysis [2
.Protein markers are often pre-stained with covalently bound chromophore to be visible to the user during and after the electrophoresis. The addition of chromophore alters the charge and size of the individual proteins in the bands of the protein marker. Thus the migration property of these bands would vary in diﬀerent electrophoresis conditions such as the use of diﬀerent buﬀer systems and matrix compositions. Therefore, these bands needs to be calibrated against the consented native proteins of natural source (e.g. Myosin, β-Galactosidase, Phosphorylase b, BSA, Lactate dehydrogenase etc.). Commercially available native protein markers include the Mark12™ unstained Standard (MK12) and the NativeMark™ unstained protein standard that are often used to calibrate the migration property of pre-stained protein markers as shown in Figure 2.
The ExcelBand™ 3-color Pre-Stained Protein Ladder Broad Range PM2700 and PM5200 with a range from 5kDa to 245kDa were calibrated against MK12 and the migration results under diﬀerent concentrations of SDS-PAGE showed a consistent curve that demonstrates the speciﬁcity of the SMOBIO ExcelBand™ PMs.
Protein markers are convenient tools for determining the approximate molecular weight of proteins. However, the complexity of the protein intrinsic properties, which present higher variability in their migration property, can result in a wildly misrepresented molecular weight while using standard SDS-PAGE analysis.
Varying concentrations of SDS-PAGE also exhibited a slightly diﬀerent protein migration whether it be the naturally sourced protein markers or the ExcelBand™ protein markers. In conclusion, ExcelBand™ has been calibrated to mimic the migration rate of native sourced proteins to minimize the possible bias cause by SDS-PAGE. For a precise determination of protein MW, analytic methods other than SDS-PAGE (e.g. MASS) are suggested.
Fig 2. Migration properties of the SMOBIO’s ExcelBand™ protein markers against the Mark12™ unstained standard showed high ﬁdelity and mirrored the migration of native-nature sourced proteins in SDS-PAGE.
Table 1. Comparative study of various amino acids their molecular weights, charge (polarity) and hydrophobicity.
1.Rath, A., et al., Detergent binding explains anomalous SDS-PAGE migration of membrane proteins. Proc Natl Acad Sci U S A, 2009. 106(6): p. 1760-5. 2.Habchi, J., et al., Structural disorder within Henipavirus nucleoprotein and phosphoprotein: from predictions to experimental assessment. PLoS One, 2010. 5(7): p. e11684.