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

Order Novex® Protein Gels

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

Table 1

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
Table 2

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
Table 3

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
Table 4

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.