Calibrating Protein Molecular Weight
The molecular weight of a protein can be determined based on its relative mobility, by constructing a standard curve using proteins of known molecular weights.
The protein mobility in SDS-PAGE gels is dependent on:
- Length of the protein in its fully denatured state
- Extent and types of protein glycosylation
- SDS-PAGE buffer systems
- Secondary structure of the protein
The same molecular weight standard may have slightly different mobility, resulting in different apparent molecular weight when run in different SDS-PAGE buffer systems. If you are using the Novex® protein molecular weight standards, use the apparent molecular masses of these standards in the gels listed in Tables 5 through 8 to determine an apparent molecular weight of your protein.
Apparent molecular masses of Mark 12™ Unstained Standards and Novex® Sharp Prestained Protein Standards under various gel and buffer conditions
Mark 12™ Unstained Standard | NuPAGE® (4–12%) Bis-Tris/MES | NuPAGE® (4–12%) Bis-Tris/MOPS | NuPAGE® (3–8%) Tris-Acetate |
---|---|---|---|
Myosin | 200 kDa | 200 kDa | 200 kDa |
β-Galactosidase | 116.3 kDa | 116.3 kDa | 116.3 kDa |
Phosphorylase B | 97.4 kDa | 97.4 kDa | 97.4 kDa |
Bovine serum albumin | 66.3 kDa | 66.3 kDa | 66.3 kDa |
Glutamic dehydrogenase | 55.4 kDa | 55.4 kDa | 55.4 kDa |
Lactate dehydrogenase | 36.5 kDa | 36.5 kDa | 36.5 kDa |
Carbonic anhydrase | 31 kDa | 31 kDa | 31 kDa |
Trypsin inhibitor | 21.5 kDa | 21.5 kDa | 21.5 kDa |
Lysozyme | 14.4 kDa | 14.4 kDa | 14.4 kDa |
Aprotinin | 6 kDa | 6 kDa | NA |
Insulin B chain | 3.5 kDa | NA | NA |
Insulin A chain | 2.5 kDa | NA | NA |
Novex® Sharp Prestained Protein Standard | NuPAGE® (4–12%) Bis-Tris/MES | NuPAGE® (4–12%) Bis-Tris/MOPS | NuPAGE® (3–8%) Tris-Acetate |
Band 1 | 260 kDa | 260 kDa | 260 kDa |
Band 2 | 160 kDa | 160 kDa | 160 kDa |
Band 3 | 110 kDa | 110 kDa | 110 kDa |
Band 4 | 80 kDa | 80 kDa | 80 kDa |
Band 5 | 60 kDa | 60 kDa | 60 kDa |
Band 6 | 50 kDa | 50 kDa | T50 kDa |
Band 7 | 40 kDa | 40 kDa | 40 kDa |
Band 8 | 30 kDa | 30 kDa | 30 kDa |
Band 9 | 20 kDa | 20 kDa | NA |
Band 10 | 15 kDa | 15 kDa | NA |
Band 11 | 10 kDa | 10 kDa | NA |
Band 12 | 3.5 kDa | NA | NA |
Apparent molecular masses of SeeBlue® and SeeBlue® Plus2 Prestained Protein Standards under various gel and buffer conditions
SeeBlue® Prestained Standard | NuPAGE® (4–12%) Bis-Tris/MES |
NuPAGE® (4–12%) Bis-Tris/MOPS |
NuPAGE® (3–8%) Tris-Acetate |
---|---|---|---|
Myosin | 188 kDa | 191 kDa | 210 kDa |
BSA | 62 kDa | 64 kDa | 71 kDa |
Glutamic dehydrogenase | 49 kDa | 51 kDa | 55 kDa |
Alcohol dehydrogenase | 38 kDa | 39 kDa | 41 kDa |
Carbonic anhydrase | 28 kDa | 28 kDa | NA |
Myoglobin | 18 kDa | 19 kDa | NA |
Lysozyme | 14 kDa | 14 kDa | NA |
Aprotinin | 6 kDa | NA | NA |
Insulin | 3 kDa | NA | NA |
SeeBlue® Plus2 Prestained Standard |
NuPAGE® (4–12%) Bis-Tris/MES |
NuPAGE® (4–12%) Bis-Tris/MOPS |
NuPAGE® (3–8%) Tris-Acetate |
Myosin | 188 kDa | 191 kDa | 210 kDa |
Phosphorylase B | 98 kDa | 97 kDa | 111 kDa |
BSA | 62 kDa | 64 kDa | 71 kDa |
Glutamic dehydrogenase | 49 kDa | 51 kDa | 55 kDa |
Alcohol dehydrogenase | 38 kDa | 39 kDa | 41 kDa |
Carbonic anhydrase | 28 kDa | 28 kDa | NA |
Myoglobin | 17 kDa | 19 kDa | NA |
Lysozyme | 14 kDa | 14 kDa | NA |
Aprotinin | 6 kDa | NA | NA |
Insulin | 3 kDa | NA | NA |
Apparent molecular masses of Novex® Sharp Prestained Protein Standards and Mark 12™ Unstained Standards on Tris-glycine and Tricine gels
Novex® Sharp Prestained Protein Standard | Tris-glycine gels (4–20%) | Tricine gels (10–20%) |
---|---|---|
Band 1 | 260 kDa | 260 kDa |
Band 2 | 160 kDa | 160 kDa |
Band 3 | 110 kDa | 110 kDa |
Band 4 | 80 kDa | 80 kDa |
Band 5 | 60 kDa | 60 kDa |
Band 6 | 50 kDa | 50 kDa |
Band 7 | 40 kDa | 40 kDa |
Band 8 | 30 kDa | 30 kDa |
Band 9 | 20 kDa | 20 kDa |
Band 10 | 15 kDa | 15 kDa |
Band 11 | 10 kDa | 10 kDa |
Band 12 | NA | 3.5 kDa |
Mark 12™ Unstained Standard | Tris-glycine gels (4–20%) | Tricine gels (10–20%) |
Myosin | 200 kDa | 200 kDa |
ϐ-Galactosidase | 116.3 kDa | 116.3 kDa |
Phosphorylase B | 97.4 kDa | 97.4 kDa |
Bovine serum albumin | 66.3 kDa | 66.3 kDa |
Glutamic dehydrogenase | 55.4 kDa | 55.4 kDa |
Lactate dehydrogenase | 36.5 kDa | 36.5 kDa |
Carbonic anhydrase | 31 kDa | 31 kDa |
Trypsin inhibitor | 21.5 kDa | 21.5 kDa |
Lysozyme | 14.4 kDa | 14.4 kDa |
Aprotinin | 6 kDa | 6 kDa |
Insulin B chain | Unresolved insulin | 3.5 kDa |
Insulin A chain | 2.5 kDa |
Apparent molecular masses of SeeBlue® and SeeBlue® Plus2 Prestained Protein Standards on Tris-glycine and Tricine gels
SeeBlue® Prestained Standard | Tris-glycine gel (4–20%) | Tricine gels (10–20%) |
---|---|---|
Myosin | 250 kDa | 210 kDa |
BSA | 98 kDa | 78 kDa |
Glutamic dehydrogenase | 64 kDa | 55 kDa |
Alcohol dehydrogenase | 50 kDa | 45 kDa |
Carbonic anhydrase | 36 kDa | 34 kDa |
Myoglobin | 30 kDa | 23 kDa |
Lysozyme | 16 kDa | 16 kDa |
Aprotinin | 6 kDa | 7 kDa |
Insulin | 4 kDa | 4 kDa |
SeeBlue® Plus2 Prestained Standard | Tris-glycine gels (4–20%) | Tricine gels (10–20%) |
Myosin | 250 kDa | 210 kDa |
Phosphorylase B | 148 kDa | 105 kDa |
BSA | 98 kDa | 78 kDa |
Glutamic acid dehydrogenase | 64 kDa | 55 kDa |
Alcohol dehydrogenase | 50 kDa | 45 kDa |
Carbonic anhydrase | 36 kDa | 34 kDa |
Myoglobin | 22 kDa | 17 kDa |
Lysozyme | 16 kDa | 16 kDa |
Aprotinin | 6 kDa | 7 kDa |
Insulin | 4 kDa | 4 kDa |
Protein Secondary Structure
When using SDS-PAGE for molecular weight determination, slight deviations from the calculated molecular weight of a protein (calculated from the known amino acid sequence) can occur due to the retention of varying degrees of secondary structure in the protein, even in the presence of SDS. This phenomenon is observed in highly organized secondary structures (collagens, histones, or highly hydrophobic membrane proteins) and in peptides, where the effect of local secondary structure becomes magnified relative to the total size of the peptide.4 kDa
Buffer Systems
Slight differences in protein mobilities also occur when the same proteins are run in different SDS-PAGE buffer systems. Each SDS-PAGE buffer system has a different pH, which affects the charge of a protein and its binding capacity for SDS. The degree of change in protein mobility is usually small in natural proteins but more pronounced with “atypical” or chemically modified proteins, such as prestained standards.